Compositions and methods for robust dynamic metabolic control

ABSTRACT

The present disclosure provides compositions and methods for rapid production of chemicals in genetically engineered microorganisms in a large scale. Also provided herein is a high-throughput metabolic engineering platform enabling the rapid optimization of microbial production strains. The platform, which bridges a gap between current in vivo and in vitro bio-production approaches, relies on dynamic minimization of the active metabolic network.

CROSS-REFERENCE

This application is a continuation of U.S. Application Ser. No.16/487,542, filed Aug. 21, 2019, which is a National Stage Entry ofPCT/US 18/19040, filed Feb. 21, 2018 which claims the benefit of U.S.Provisional Application No. 62/461,436, filed Feb. 21, 2017, whichapplication is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Federal Grant Nos.HRI 1-14-C-0075 awarded by DOD/DARPA, 12043956 and N00014-16-1-2558awarded by NAVY/ONR, and 1445726 awarded by NSF. The Government hascertain rights to this invention.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Feb. 21, 2018, is named52240_702_601_SL.txt and is 81,697 bytes in size.

BACKGROUND OF THE INVENTION

Biotechnology-based fermentation processes have been successfullydeveloped to produce everything from biologics and small moleculetherapies to specialty, bulk and commodity chemicals, and even nextgeneration biofuels. These processes have made rapid advancements inrecent years due to technology developments in the fields offermentation science and synthetic biology, as well as metabolic andenzyme engineering. Despite these substantial advances, most successfulexamples of rational and directed engineering approaches have alsogreatly relied on numerous and often lengthy cycles of trial and error.The present disclosure provides a strategy that simultaneously reducesthe complexity of the problem (as well as the size of the relevantdesign space), while also minimizing metabolic responses toenvironmental conditions, increasing robustness and scalability ofengineered strains.

SUMMARY OF THE INVENTION

The present disclosure provides, in part, a high-throughput engineeringplatform that enables the rapid development of microbial productionstrains.

In one aspect, the present disclosure provides a cell for generating aproduct, wherein the cell comprises: a heterologous polynucleotide forcontrolled reduction of expression of an enzyme of a metabolic pathway,wherein the controlled reduction of expression of the enzyme induces astationary phase of the cell; and a heterologous productionpolynucleotide for mediating controlled increase in expression of aproduction enzyme for generation of the product; wherein a rate ofproduction of the product during the stationary phase is reduced less inresponse to a change of an environmental condition as compared to a celllacking the enzyme.

In some embodiments, the heterologous polynucleotide reduces fluxthrough the metabolic pathway. In some embodiments, the enzyme isselected from the group consisting of enoyl-ACP/CoA reductase,glucose-6-phosphate dehydrogenase, lipoamide dehydrogenase, citratesynthase, soluble transhydrogenase, and NADH-dependentglyceraldehyde-3-phosphate dehydrogenase. In some embodiments, theproduction enzyme is selected from the group consisting ofNADPH-dependent alanine dehydrogenase, an alanine exporter, andNADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. In someembodiments, the change of an environmental condition comprisesincreasing or decreasing a concentration of a sugar in a culture mediumcontacting the cell. In some embodiments, the sugar is glucose. In someembodiments, the change of an environmental condition comprisesincreasing or decreasing oxygenation of a culture medium contacting thecell. In some embodiments, the product comprises 3-hydroxypropionicacid.

In some embodiments, the product comprises an amino acid. In someaspects, the amino acid comprises alanine. In some aspects, the cell isgrown in a culture, and a rate of production of the alanine by theculture is at least 0.5 g/L/hour. In some aspects, the rate ofproduction of the alanine is at least 1.0 g/L/hour. In some aspects, therate of production of the alanine is at least 1.5 g/L/hour. In someaspects, the rate of production of the alanine is at least 1.6 g/L/hour.In some aspects, the culture produces at least 80 g/L of the alanine. Insome aspects, the culture produces at least 100 g/L of the alanine. Insome aspects, the culture produces at least 120 g/L of the alanine. Insome aspects, the culture produces at least 140 g/L of the alanine. Insome aspects, the production polynucleotide encodes an alanine exporter.In some aspects, the alanine exporter is alaE.

In some embodiments, the product comprises mevalonic acid. In someembodiments, the cell is grown in a culture, and a rate of production ofthe mevalonic acid by the culture is at least 0.5 g/L/hour. In someembodiments, the rate of production of the mevalonic acid is at least1.0 g/L/hour. In some embodiments, the rate of production of themevalonic acid is at least 1.2 g/L/hour. In some embodiments, the rateof production of the mevalonic acid is at least 1.25 g/L/hour. In someaspects, the cell is grown in a culture, and the culture produces atleast 50 g/L of the mevalonic acid. In some embodiments, the cultureproduces at least 70 g/L of the mevalonic acid. In some embodiments, theculture produces at least 90 g/L of the mevalonic acid. In someembodiments, the culture produces at least 95 g/L of the mevalonic acid.In some embodiments, the heterologous polynucleotide is selected fromthe group consisting of: a silencing polynucleotide for repressingtranscription of a gene encoding the enzyme; and a degradationpolynucleotide for mediating cellular degradation of the enzyme.

In some aspects, the heterologous polynucleotide comprises a silencingpolynucleotide, and the silencing polynucleotide comprises a guide RNA(gRNA) comprising a gRNA sequence that recognizes a promoter of a geneencoding the enzyme. In some aspects, the heterologous polynucleotideencodes a CRISPR enzyme, and the CRISPR enzyme specifically binds to thepromoter sequence when bound to the gRNA. In some aspects, the CRISPRenzyme is catalytically inactive. In some aspects, the heterologouspolynucleotide comprises a degradation polynucleotide, wherein thedegradation polynucleotide comprises a sequence encoding a degradationtag, wherein the degradation tag mediates degradation of the enzyme. Insome embodiments, expression of the heterologous polynucleotide isregulated by phosphate availability in the cell. In some embodiments,expression of the production polynucleotide is regulated by phosphateavailability in the cell. In some embodiments, the cell is an E. colicell.

In another aspect, disclosed herein is a method comprising: culturingindependently a plurality of strains of a cell, wherein each straincomprises (i) a heterologous polynucleotide for mediating controlledreduction of expression of an enzyme of a metabolic pathway, wherein thecontrolled reduction of expression of the enzyme induces a stationaryphase of the cell; and (ii) a heterologous production polynucleotide formediating controlled increase in expression of a production enzyme forgeneration of the product; wherein each strain of the plurality ofstrains differs from another strain in a sequence of at least one of theheterologous polynucleotide or the heterologous productionpolynucleotide; growing the plurality of strains to stationary phase;and selecting a strain of the plurality of strains based on a level ofthe product produced by the selected strain during the stationary phase.

In some embodiments, the method comprises determining the level of theproduct. In some embodiments, the method comprises growing the selectedstrain. In some embodiments, the selected strain is grown in abioreactor. In some embodiments, a culture medium comprising theselected strain has a volume of at least 500 ml. In some embodiments,the culture medium has a volume of at least 1 L. In some embodiments,the heterologous polynucleotide is selected from the group consistingof: a silencing polynucleotide for repressing transcription of a geneencoding the enzyme; and a degradation polynucleotide for mediatingcellular degradation of the enzyme. In some embodiments, a first andsecond strain of the plurality of strains comprises a silencingpolynucleotide. In some embodiments, the silencing polynucleotidecomprises a guide RNA (gRNA) comprising a gRNA sequence that recognizesa promoter sequence of a gene encoding the enzyme. In some embodiments,the gRNA sequence differs between the first and second strains. In someembodiments, the first and second strain of the plurality of strainscomprise a degradation polynucleotide. In some embodiments, thedegradation polynucleotide differs between the first and second strains.In some embodiments, the enzyme is selected from the group consisting ofenoyl-ACP/CoA reductase, glucose-6-phosphate dehydrogenase, lipoamidedehydrogenase, citrate synthase, soluble transhydrogenase, andNADH-dependent glyceraldehyde-3-phosphate dehydrogenase. In someembodiments, the production enzyme is selected from the group consistingof NADPH-dependent alanine dehydrogenase, an alanine exporter, andNADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. In someembodiments, the product is selected from the group consisting ofmevalonic acid, 3-hydroxypropionic acid, and an amino acid.

In some embodiments, the product is an amino acid and the amino acid isalanine. In some embodiments, the cell of the selected strain a rate ofproduction of the product during the stationary phase is reduced less inresponse to a change of an environmental condition as compared to a celllacking the heterologous polynucleotide. In some embodiments, the changeof an environmental condition comprises a change in concentration of asugar of a culture medium contacting the cell. In some embodiments, thechange of an environmental condition comprises a change in oxygenationof a culture medium contacting the cell.

In another aspect, disclosed herein is a method of generating a cellularproduct comprising: culturing a heterologous cell in a culture medium,wherein the heterologous cell comprises: (i) a heterologouspolynucleotide for mediating controlled reduction of expression of anenzyme of a metabolic pathway, wherein the controlled reduction ofexpression of the enzyme induces a stationary phase of the cell; and(ii) a heterologous production polynucleotide for mediating controlledincrease in expression of a production enzyme for generation of theproduct; wherein a rate of production of the product during thestationary phase is reduced less in response to a change of anenvironmental condition as compared to a cell lacking the enzyme.

In one embodiment, the method further comprises changing theenvironmental condition. In one embodiment, the environmental conditioncomprises a concentration of a sugar of the culture medium, and changingthe environmental condition comprises increasing or decreasing theconcentration. In some embodiments, the sugar is glucose. In someembodiments, the environmental condition comprises an oxygenconcentration of the culture medium, and changing the environmentalcondition comprises increasing or decreasing the oxygen concentration.In some embodiments, the culturing is performed in a bioreactor. In someembodiments, the culture medium has a volume of at least 500 ml. In someembodiments, the culture medium has a volume of at least 1 L. In someembodiments, the product comprises 3-hydroxypropionic acid. In someembodiments, the product comprises an amino acid. In some embodiments,the amino acid comprises alanine. In some embodiments, the rate ofproduction of the alanine is at least 0.5 g/L/hour. In some embodiments,the rate of production of the alanine is at least 1.0 g/L/hour. In someembodiments, the rate of production of the alanine is at least 1.5g/L/hour. In some embodiments, the rate of production of the alanine isat least 1.6 g/L/hour. In some embodiments, the productionpolynucleotide encodes an alanine exporter. In some embodiments, thealanine exporter is alaE.

In some embodiments, the product comprises mevalonic acid. In someembodiments, the rate of production of the mevalonic acid is at least0.5 g/L/hour. In some embodiments, the rate of production of themevalonic acid is at least 1.0 g/L/hour. In some embodiments, the rateof production of the mevalonic acid is at least 1.2 g/L/hour. In someembodiments, the rate of production of the mevalonic acid is at least1.25 g/L/hour. In some embodiments, the heterologous polynucleotide isselected from the group consisting of: a silencing polynucleotide forrepressing transcription of a gene encoding the enzyme; and adegradation polynucleotide for mediating cellular degradation of theenzyme. In some embodiments, the heterologous polynucleotide comprises asilencing polynucleotide, and the silencing polynucleotide comprises aguide RNA (gRNA) comprising a gRNA sequence that recognizes a promotersequence of a gene encoding the enzyme. In some embodiments, theheterologous polynucleotide encodes a CRISPR enzyme, wherein the CRISPRenzyme specifically binds to the promoter sequence when bound to thegRNA. In some embodiments, the CRISPR enzyme is catalytically inactive.In some embodiments, the heterologous polynucleotide comprises adegradation polynucleotide, wherein the degradation polynucleotidecomprises a sequence encoding a degradation tag, wherein the degradationtag mediates degradation of the enzyme. In some embodiments, theexpression of the heterologous polynucleotide is regulated by phosphateavailability in the cell. In some embodiments, the expression of theproduction polynucleotide is regulated by phosphate availability in thecell. In some embodiments, the cell is an E. coli cell.

In another aspect, disclosed herein is a cell for production of alanine,wherein the cell comprises: (i) a heterologous polynucleotide forcontrolled reduction of expression of an enzyme of a metabolic pathway,wherein the enzyme is selected from the group consisting ofenoyl-ACP/CoA reductase, glucose-6-phosphate dehydrogenase, lipoamidedehydrogenase (lpd), citrate synthase (gltA), soluble transhydrogenase,and NADH-dependent glyceraldehyde-3-phosphate dehydrogenase; and (ii) analanine exporter, wherein the alanine exporter is expressed at increasedlevels as compared to a wildtype cell.

In some embodiments, the alanine exporter is encoded by an alaE gene. Insome embodiments, the controlled reduction of expression of the enzymeinduces a stationary phase of the cell. In some embodiments, the cellfurther comprises a heterologous production polynucleotide forcontrolled increase in expression of a production enzyme for generationof the alanine. In some embodiments, the production enzyme is selectedfrom the group consisting of NADPH-dependent alanine dehydrogenase andNADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. In someembodiments, the heterologous polynucleotide is selected from the groupconsisting of: a silencing polynucleotide for mediating transcriptionalrepression of a gene encoding the enzyme; and a degradationpolynucleotide for mediating cellular degradation of the enzyme. In someembodiments, the heterologous polynucleotide comprises a silencingpolynucleotide, and the silencing polynucleotide comprises a guide RNA(gRNA) comprising a gRNA sequence that recognizes a promoter sequence ofa gene encoding the enzyme. In some embodiments, the polynucleotidefurther encodes a CRISPR enzyme, wherein the CRISPR enzyme specificallybinds to the promoter sequence when bound to the gRNA. In someembodiments, the CRISPR enzyme is catalytically inactive. In someembodiments, the heterologous polynucleotide comprises a degradationpolynucleotide, wherein the degradation polynucleotide comprises asequence encoding a degradation tag, wherein the degradation tagmediates degradation of the enzyme. In some embodiments, thepolynucleotide is regulated by phosphate availability in the cell. Insome embodiments, the production polynucleotide is regulated byphosphate availability in the cell. In some embodiments, the cell is anE. coli cell.

In some embodiments, a culture comprises the cell. In some embodiments,a rate of production of the alanine by the culture is at least 0.5g/L/hour. In some embodiments, a rate of production of the alanine bythe culture is at least 1.0 g/L/hour. In some embodiments, a rate ofproduction of the alanine by the culture is at least 1.5 g/L/hour. Insome embodiments, a rate of production of the alanine by the culture isat least 1.6 g/L/hour. In some embodiments, the culture produces atleast 100 g/L of the alanine. In some embodiments, the culture producesat least 120 g/L of the alanine. In some embodiments, the cultureproduces at least 140 g/L of the alanine.

In some aspects, disclosed herein is a method of production of alaninecomprising growing in a culture medium a cell comprising (i) aheterologous polynucleotide for controlled reduction of expression of aenzyme of a metabolic pathway, wherein the enzyme is selected from thegroup consisting of enoyl-ACP/CoA reductase, glucose-6-phosphatedehydrogenase, lipoamide dehydrogenase, citrate synthase, solubletranshydrogenase, and NADH-dependent glyceraldehyde-3-phosphatedehydrogenase; and (ii) an alanine exporter, wherein the alanineexporter is expressed at increased levels as compared to a wildtypecell.

In some embodiments, the controlled reduction of expression of theenzyme induces a stationary phase of the cell. In some embodiments, themethod further comprises decreasing an oxygenation level or a sugarconcentration of the culture medium during the stationary phase, whereina rate of production of the cellular product is reduced less in responseto the decreasing as compared to a cell lacking the heterologouspolynucleotide. In some embodiments, the sugar is glucose. In someembodiments, the alanine exporter is encoded by an alaE gene. In someembodiments, the cell further comprises a heterologous productionpolynucleotide for controlled increase in expression of a productionenzyme for generation of the alanine. In some embodiments, theproduction enzyme is selected from the group consisting of:NADPH-dependent alanine dehydrogenase and NADPH-dependentglyceraldehyde-3-phosphate dehydrogenase. In some embodiments, theheterologous polynucleotide is selected from the group consisting of: asilencing polynucleotide for mediating transcriptional repression of agene encoding the enzyme; and a degradation polynucleotide for mediatingcellular degradation of the enzyme. In some embodiments, theheterologous polynucleotide comprises a silencing polynucleotide, andthe silencing polynucleotide comprises a guide RNA (gRNA) comprising agRNA sequence that recognizes a promoter sequence of a gene encoding theenzyme. In some embodiments, the heterologous polynucleotide encodes aCRISPR enzyme, wherein the CRISPR enzyme specifically binds to thepromoter sequence when bound to the gRNA. In some embodiments, theCRISPR enzyme is catalytically inactive. In some embodiments, theheterologous polynucleotide comprises a degradation polynucleotide,wherein the degradation polynucleotide comprises a sequence encoding adegradation tag, wherein the degradation tag mediates degradation of theenzyme.

In some embodiments, the expression of the heterologous polynucleotideis regulated by phosphate availability in the cell. In some embodiments,the production polynucleotide is regulated by phosphate availability inthe cell. In some embodiments, the cell is an E. coli cell. In someembodiments, a rate of production of the alanine is at least 0.5g/L/hour. In some embodiments, a rate of production of the alanine is atleast 1.0 g/L/hour. In some embodiments, a rate of production of thealanine is at least 1.5 g/L/hour. In some embodiments, a rate ofproduction of the alanine is at least 1.6 g/L/hour.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A depicts an overview of dynamic metabolic control in 2-stagefermentations.

FIG. 1B depicts strain and bioprocess optimization.

FIGS. 2A-D depict an example of implementation of 2-stage SyntheticMetabolic Valves (SMVs) in E. coli.

FIGS. 3A-K depict an example of alanine production in E. coli utilizing2-stage dynamic control.

FIGS. 4A-F depict example robustness comparison between 2-stage andgrowth associated approaches.

FIGS. 5A-J depict example comparisons of “Valve” and growth associatedalanine production in micro-fermentations and 1 L fermentation.

FIG. 6A-H depict an example of mevalonate production in E. coliutilizing 2-stage dynamic control.

FIG. 7 depicts an example of phosphate depletion promotercharacterization.

FIG. 8 depicts an example of insulated phosphate depletion promotercharacterization.

FIG. 9 depicts an example of insulated constitutive promotercharacterization.

FIG. 10 depicts an example of metabolic modeling results for optimal3-HP flux in two stage fermentations.

FIG. 11 depicts examples of chromosomal modifications.

FIG. 12 depicts an example of average maximal growth rates of startinghost strains in 1 L FGM10 minimal medium fermentations, n=2.

FIG. 13A-E depict examples of distribution of glucose utilized duringthe growth phase of starting host strains in 1 L standard minimal mediumfermentations.

FIG. 14 depicts pCASCADE-control plasmid construction scheme.

FIGS. 15A-B depict pCASCADE construction scheme.

FIGS. 16A-C depict an overview of micro-fermentation process.

FIG. 17 depicts micro-fermentation for L-alanine production usingdifferent insulated phosphate promoters in DLF_0025 strain.

FIG. 18 depicts Heatmap for L-alanine production by gapN/gapA strains.

FIGS. 19A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 20A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 21A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 22A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 23A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 24A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 25A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 26A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 27A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 28A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 29A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 30A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 31A-D depict alanine production in response to different OTR andglucose concentration in micro-fermentation for 4 strains evaluated forrobustness.

FIG. 32 depicts alanine production in response to different OTR andglucose concentration in micro-fermentation for one strain evaluated forrobustness.

FIGS. 33A-B depict growth profile for all valve and growth associatedstrains at 1 L scale evaluated in this paper.

FIG. 34 depicts specific Productivity (SP) comparison for strain withhighest mevalonate titer from literature and mevalonate strain 1evaluated in this work.

FIG. 35 depicts alanine standard curve from MS measurement. Average andstandard deviation for mass spec response from triplicate standardmeasurement were plotted.

FIGS. 36A-B depict glucose and ethanol standard curves from RImeasurement.

FIG. 37 depicts 3-Hydroxypropionic acid standard curve from TUVmeasurement.

FIGS. 38A-D depict TUV standard curves for L-alanine, D-alanine,mevalonic acid, and mevalonolactone.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in the specification and the claims, the singular forms “a,”“an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to an “expressionvector” includes a single expression vector as well as a plurality ofexpression vectors, either the same (e.g., the same operon) ordifferent; reference to “microorganism” includes a single microorganismas well as a plurality of microorganisms; and the like.

As used herein, “reduced enzymatic activity,” “reducing enzymaticactivity.” and the like is meant to indicate that a microorganismcell's, or an isolated enzyme, exhibits a lower level of activity thanthat measured in a comparable cell of the same species or its nativeenzyme. That is, enzymatic conversion of the indicated substrate(s) toindicated product(s) under known standard conditions for that enzyme isat least 10, at least 20, at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, or at least 90 percent less than theenzymatic activity for the same biochemical conversion by a native(non-modified) enzyme under a standard specified condition. This termalso can include elimination of that enzymatic activity. A cell havingreduced enzymatic activity of an enzyme can be identified using anymethod known in the art. For example, enzyme activity assays can be usedto identify cells having reduced enzyme activity. See, for example,Enzyme nomenclature, Academic Press, Inc., New York 2007.

The term “heterologous DNA,” “heterologous nucleic acid sequence,” andthe like as used herein refers to a nucleic acid sequence wherein atleast one of the following is true: (a) the sequence of nucleic acidsforeign to (i.e., not naturally found in) a given host microorganism;(b) the sequence may be naturally found in a given host microorganism,but in an unnatural (e.g., greater than expected) amount; or (c) thesequence of nucleic acids comprises two or more subsequences that arenot found in the same relationship to each other in nature. For example,regarding instance (c), a heterologous nucleic acid sequence that isrecombinantly produced will have two or more sequences from unrelatedgenes arranged to make a new functional nucleic acid, such as anonnative promoter driving gene expression.

The term “synthetic metabolic valve,” and the like as used herein refersto either the use of controlled proteolysis, gene silencing or thecombination of both proteolysis and gene silencing to alter metabolicfluxes.

The term “heterologous” is intended to include the term “exogenous” asthe latter term is generally used in the art. With reference to the hostmicroorganism's genome prior to the introduction of a heterologousnucleic acid sequence, the nucleic acid sequence that codes for theenzyme is heterologous (whether or not the heterologous nucleic acidsequence is introduced into that genome).

As used herein, the term “gene disruption,” or grammatical equivalentsthereof (and including “to disrupt enzymatic function,” “disruption ofenzymatic function.” and the like), is intended to mean a geneticmodification to a microorganism that renders the encoded gene product ashaving a reduced polypeptide activity compared with polypeptide activityin or from a microorganism cell not so modified. The geneticmodification can be, for example, deletion of the entire gene, deletionor other modification of a regulatory sequence required fortranscription or translation, deletion of a portion of the gene whichresults in a truncated gene product (e.g., enzyme) or by any of variousmutation strategies that reduces activity (including reducing activitiesto no detectable activity level) the encoded gene product. A disruptionmay broadly include a deletion of all or part of the nucleic acidsequence encoding the enzyme, and also includes, but is not limited toother types of genetic modifications, e.g., introduction of stop codons,frame shift mutations, introduction or removal of portions of the gene,and introduction of a degradation signal, those genetic modificationsaffecting mRNA transcription levels and/or stability, and altering thepromoter or repressor upstream of the gene encoding the enzyme.

Bio-production or fermentation, as used herein, may be aerobic,microaerobic, or anaerobic.

When the genetic modification of a gene product, e.g., an enzyme, isreferred to herein, including the claims, it is understood that thegenetic modification is of a nucleic acid sequence, such as or includingthe gene, that normally encodes the stated gene product, e.g., theenzyme.

As used herein, the term “metabolic flux” and the like refers to changesin metabolism that lead to changes in product and/or byproductformation, including production rates, production titers and productionyields from a given substrate.

Species and other phylogenic identifications are according to theclassification known to a person skilled in the art of microbiology.

Enzymes are listed here within, with reference to a Universal ProteinResource (Uniprot) identification number, which would be well known toone skilled in the art (Uniprot is maintained by and available throughthe UniProt Consortium).

Where methods and steps described herein indicate certain eventsoccurring in certain order, those of ordinary skill in the art willrecognize that the ordering of certain steps may be modified and thatsuch modifications are in accordance with the variations of theinvention. Additionally, certain steps may be performed concurrently ina parallel process when possible, as well as performed sequentially.

The meaning of abbreviations is as follows: “C” means Celsius or degreesCelsius, as is clear from its usage, DCW means dry cell weight, “s”means second(s), “min” means minute(s), “h”, “hr,” or “hrs” meanshour(s), “psi” means pounds per square inch, “nm” means nanometers, “d”means day(s), “μL” or “uL” or “ul” means microliter(s), “mL” meansmilliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” meansnanometers, “mM” means millimolar, “μM” or “uM” means micromolar, “M”means molar, “mmol” means millimole(s), “μmol” or “uMol” meansmicromole(s), “g” means gram(s), “μg” or “ug” means microgram(s) and“ng” means nanogram(s), “PCR” means polymerase chain reaction, “OD”means optical density. “OD₆₀₀” means the optical density measured at aphoton wavelength of 600 nm, “kDa” means kilodaltons, “g” means thegravitation constant, “bp” means base pair(s). “kbp” means kilobasepair(s), “% w/v” means weight/volume percent, “% v/v” meansvolume/volume percent, “IPTG” meansisopropyl-μ-D-thiogalactopyranoiside, “aTc” means anhydrotetracycline,“RBS” means ribosome binding site, “rpm” means revolutions per minute,“HPLC” means high performance liquid chromatography, and “GC” means gaschromatography.

Overview

Provided herein is a high-throughput metabolic engineering platformenabling the rapid optimization of microbial production strains. Theplatform, which bridges a gap between current in vivo and in vitrobio-production approaches, relies on dynamic minimization of the activemetabolic network. Dynamic metabolic network minimization can beaccomplished using combinations of CRISPR interference and controlledproteolysis to reduce the activity of multiple enzymes in essentialcentral metabolism. Minimization can be implemented in the context ofstandardized 2-stage bio-processes. This approach not only can result ina design space with greatly reduced complexity, but also in increasedmetabolic fluxes and production rates as well as in strains which arerobust to environmental conditions. Robustness can lead to predictablescalability from high-throughput small-scale screens, or“micro-fermentations”, to fully instrumented bioreactors. Predictivehigh-throughput approaches may be critical for metabolic engineeringprograms to truly take advantage of the rapidly increasing throughputand decreasing costs of synthetic biology. The examples provided hereinhave not only demonstrated proof of principle for this approach in thecommon industrial microbe: E. coli, and has validated this approach withthe rapid optimization of E. coli strains producing two importantindustrial chemicals: alanine and mevalonic acid, at commerciallymeaningful rates, titers (147 g/L and 97 g/L, respectively), and yields.

Also provided herein are systems and methods to rapidly optimize amicroorganism for chemical productions in a high-throughput fashion.

Also provided herein are microorganisms that can be used with thedisclosed platform and/or methods for chemical productions.

Synthetic Metabolic Valves (SMVs)

The current disclosure describes the construction of synthetic metabolicvalves (SMVs) comprising one or more or a combination of the following:controlled gene silencing and controlled proteolysis. It is appreciatedthat one well skilled in the art is aware of several methodologies forgene silencing and controlled proteolysis.

The development of platform microbial strains that utilize SMVs candecouple growth from product formation. These strains enable the dynamiccontrol of metabolic pathways, including those that when altered havenegative effects on microorganism growth. Dynamic control overmetabolism is accomplished via a combination of methodologies includingbut not limited to transcriptional silencing and controlled enzymeproteolysis. These microbial strains are utilized in a multi-stagebioprocess encompassing as least two stages, the first stage in whichmicroorganisms are grown and metabolism can be optimized for microbialgrowth and at least one other stage in which growth can be slowed orstopped, and dynamic changes can be made to metabolism to improveproduction of desired product, such as a chemical or fuel. Thetransition of growing cultures between stages and the manipulation ofmetabolic fluxes can be controlled by artificial chemical inducers orpreferably by controlling the level of key limiting nutrients. Inaddition, genetic modifications may be made to provide metabolicpathways for the biosynthesis of one or more chemical or fuel products.Also, genetic modifications may be made to enable the utilization of avariety of carbon feedstocks including but not limited sugars such asglucose, sucrose, xylose, arabinose, mannose, and lactose, oils, carbondioxide, carbon monoxide, methane, methanol and formaldehyde.

This approach allows for simpler models of metabolic fluxes andphysiological demands during a production phase, turning a growing cellinto a stationary phase biocatalyst. These synthetic metabolic valvescan be used to turn off essential genes and redirect carbon, electronsand energy flux to product formation in a multi-stage fermentationprocess. One or more of the following enables these synthetic valves: 1)transcriptional gene silencing or repression technologies in combinationwith 2) inducible enzyme degradation and 3) nutrient limitation toinduce a stationary or non-dividing cellular state. SMVs aregeneralizable to any pathway and microbial host. These syntheticmetabolic valves allow for novel rapid metabolic engineering strategiesuseful for the production of renewable chemicals and fuels and anyproduct that can be produced via whole cell catalysis.

In various cases, one SMV can refer to the manipulation of one gene (orits protein product). The manipulation can be controlled silencing ofthe gene and/or controlled degradation of its protein product. Incertain cases, combination of SMVs can lead to improved production inyields, rate and/or robustness, which includes manipulation of two genes(or their protein products). In some cases, an engineered microorganismcomprises at least one SMV. In some cases, an engineered microorganismcomprises more than one SMV. In some cases, an engineered microorganismcomprises two, three, four, five, six, seven, eight, nine, or ten, ormore SMVs.

Method and Systems for Bio-Production

Provided herein are methods or systems for robust large scale productionof molecules from biologics and small molecule therapeutics tospecialty, bulk and commodity chemicals, and biofuels. The methods orsystems provided herein comprise using engineered microorganism whichcomprises a limited set of metabolic enzymes. In some embodiments, theengineered microorganism comprises at least one metabolic enzyme thathas reduced level or activity. In some embodiments, the engineeredmicroorganism comprises two, three, four, five, six, seven, eight, nine,or ten, or more metabolic enzymes that have reduced level or activity.The methods and systems provided herein can reduce metabolic responsesto environmental conditions and can be easily transferred from smallscale (e.g. mgs) production to large scale (e.g. kgs) production. Themethods and systems provided herein can reduce the time and costsassociated with transitioning from small scale (e.g. mgs) to large scale(e.g. kgs) production.

Within the scope of the current disclosure are genetically modifiedmicroorganism, wherein the microorganism is capable of producing aproduct derived from any key metabolic intermediate including but notlimited to malonyl-CoA, pyruvate, oxaloacetate, erthyrose-4-phosphate,xylulose-5-phosphate, alpha-ketoglutarate and citrate at a specific rateselected from the rates of greater than 0.05 g/gDCW-hr, 0.08 g/gDCW-hr,greater than 0.1 g/gDCW-hr, greater than 0.13 g/gDCW-hr. greater than0.15 g/gDCW-hr, greater than 0.175 g/gDCW-hr, greater than 0.2g/gDCW-hr, greater than 0.25 g/gDCW-hr. greater than 0.3 g/gDCW-hr,greater than 0.35 g/gDCW-hr, greater than 0.4 g/gDCW-hr, greater than0.45 g/gDCW-hr. or greater than 0.5 g/gDCW-hr.

In various embodiments, the invention includes a culture systemcomprising a carbon source in an aqueous medium and a geneticallymodified microorganism, wherein said genetically modified organism ispresent in an amount selected from greater than 0.05 gDCW/L, 0.1 gDCW/L,greater than 1 gDCW/L, greater than 5 gDCW/L, greater than 10 gDCW/L,greater than 15 gDCW/L or greater than 20 gDCW/L, such as when thevolume of the aqueous medium is selected from greater than 5 mL, greaterthan 100 mL, greater than 0.5 L, greater than 1 L, greater than 2 L,greater than 10 L, greater than 250 L, greater than 1000 L, greater than10,000 L, greater than 50,000 L, greater than 100,000 L or greater than200,000 L, and such as when the volume of the aqueous medium is greaterthan 250 L and contained within a steel vessel.

Carbon Sources

Bio-production media, which is used in the present invention withrecombinant microorganisms must contain suitable carbon sources orsubstrates for both growth and production stages. Suitable substratesmay include, but are not limited to glucose, sucrose, xylose, mannose,arabinose, oils, carbon dioxide, carbon monoxide, methane, methanol,formaldehyde and glycerol. It is contemplated that all of the abovementioned carbon substrates and mixtures thereof are suitable in thepresent invention as a carbon source(s).

Microorganism

Features as described and claimed herein may be provided in amicroorganism selected from the listing herein, or another suitablemicroorganism, that also comprises one or more natural, introduced, orenhanced product bio-production pathways. Thus, in some embodiments themicroorganism(s) comprise an endogenous product production pathway(which may, in some such embodiments, be enhanced), whereas in otherembodiments the microorganism does not comprise an endogenous productproduction pathway.

The examples describe specific modifications and evaluations to certainbacterial and fungal microorganisms. The scope of the invention is notmeant to be limited to such species, but to be generally applicable to awide range of suitable microorganisms.

Suitable host cells or host microorganisms for bio-production can beeither prokaryotic or eukaryotic. Suitable host cells or hostmicroorganisms can be bacteria such as Citrobacter, Enterobacter,Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus,Saccharomyces. Schizosaccharomyces, Zygosaccharomyces, Pichia.Kluyveromyces. Candida, Hansenula, Debaryomyces, Mucor, Torulopsis,Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, andPseudomonas. In some embodiments, a host cell or an engineered cell isE. coli. In some embodiments, a host cell or an engineered cell is S.cerevisiae.

In certain aspects, provided herein is a microorganism geneticallymodified to comprise: a production pathway comprising at least oneenzyme for the biosynthesis of a product, and a combination of multiplesynthetic metabolic valves to controllably reduce or eliminate fluxthrough multiple metabolic pathways. In some embodiments, each of themultiple synthetic metabolic valves comprises one or more genes for (i)controlled silencing of gene expression of at least one gene or (ii) thecontrolled proteolytic inactivation of at least one protein. In someembodiments, a rate of the biosynthesis of the product is increased in aproductive stationary phase upon a depletion of a nutrient, wherein thedepletion of the nutrient induces the multiple synthetic metabolicvalves. In some cases, the controlled silencing of gene expression isaccomplished by RNA interference. CRISPR interference or transcriptionalrepression. In some cases, the controlled proteolytic inactivation isaccomplished by protein cleavage by a specific protease or targeteddegradation by specific peptide tags. In some cases, the nutrient isphosphate, nitrogen, sulfur, magnesium, or a combination thereof.

In certain aspects, provided herein is a genetically modifiedmicroorganism comprising: a production pathway comprising at least oneenzyme for the biosynthesis of a product from one of the followingmetabolites: pyruvate, acetolactate, acetyl-CoA, acetoacetyl-CoA ormalonyl-CoA; and a combination of multiple synthetic metabolic valves,wherein each of the multiple synthetic metabolic valves comprises one ofa fabI, gltA, lpd, zuf or udhA gene for (i) controlled silencing of geneexpression of a corresponding one of said fabI, gltA, lpd, zwf or udhAgenes or (ii) controlled proteolytic inactivation of a protein encodedby a corresponding one of said fabI, gltA, lpd, zwf or udhA genes. Insome embodiments, a rate of the biosynthesis of the product is increasedin a productive stationary phase upon a depletion of a nutrient, whereinthe depletion of the nutrient induces the multiple synthetic metabolicvalves. In some embodiments, the product is alanine or a derivativethereof. In some embodiments, the product is mevalonate or a derivativethereof. In some embodiments, the product is malonic acid or aderivative thereof. In some embodiments, the nutrient is phosphate,nitrogen, sulfur, magnesium, or a combination thereof.

In certain aspects, provided herein is a genetically modifiedmicroorganism comprising: a production pathway to produce alanine frompyruvate; and a combination of multiple synthetic metabolic valves,wherein each of the multiple synthetic metabolic valves comprises one ofa fabI, gltA, lpd, zwf or udhA gene for (i) controlled silencing of geneexpression of a corresponding one of said fabI, gltA, lpd, zwf or udhAgenes or (ii) controlled proteolytic inactivation of a protein encodedby one of said fabI, gltA, lpd, zuf or udhA genes. In some embodiments,a rate of the biosynthesis of alanine is increased in a productivestationary phase upon a depletion of a nutrient, wherein the depletionof the nutrient induces the multiple synthetic metabolic valves. In someembodiments, the nutrient is phosphate, nitrogen, sulfur, magnesium, ora combination thereof.

In some cases, a genetically modified microorganism is a heterologouscell. In some cases, provided herein is a heterologous cell forgenerating a product. In some cases, a heterologous cell comprises anengineered valve polynucleotide for mediating controlled reduction ofexpression of a valve enzyme acting in a metabolic pathway. In certaincases, a controlled reduction of expression of a valve enzyme reducesflux through a metabolic pathway, wherein the controlled reduction ofexpression of the valve enzyme induces a stationary phase of theheterologous cell. In some cases, a heterologous cell further comprisesan engineered production polynucleotide for mediating controlledincrease in expression of a production enzyme for generation of theproduct. In some situations, a heterologous cell comprises an engineeredvalve polynucleotide for mediating controlled reduction of expression ofa valve enzyme acting in a metabolic pathway, wherein a rate ofproduction of a product during a stationary phase is reduced less inresponse to a change of an environmental condition as compared to a celllacking the controlled reduction of expression of the valve enzyme.

In some cases, provided herein is a heterologous cell for generating aproduct, wherein said cell comprises: an engineered valve polynucleotidefor mediating controlled reduction of expression of a valve enzymeacting in a metabolic pathway, wherein said controlled reduction ofexpression of said valve enzyme reduces flux through said metabolicpathway, wherein said controlled reduction of expression of said valveenzyme induces a stationary phase of said cell; and an engineeredproduction polynucleotide for mediating controlled increase inexpression of a production enzyme for generation of said product;wherein a rate of production of said product during said stationaryphase is reduced less in response to a change of an environmentalcondition as compared to a cell lacking said controlled reduction ofexpression of said valve enzyme.

In some cases, provided herein is a cell comprising a reduced expressionor activity of a valve enzyme, wherein the valve enzyme comprises anenzyme selected from the group consisting of enoyl-ACP/CoA reductase(fabI), glucose-6-phosphate dehydrogenase (zwf), lipoamide dehydrogenase(lpd), citrate synthase (gltA), soluble transhydrogenase (udhA),NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapA), and acombination thereof.

In some cases, provided herein is a cell comprising a production enzyme,wherein the production enzyme comprises an enzyme selected from thegroup consisting of NADPH-dependent alanine dehydrogenase (aid), alanineexporter (alaE), NADPH-dependent glyceraldehyde-3-phosphatedehydrogenase (gapN), and a combination thereof.

Environmental Conditions

Environmental conditions can comprise medium and culture conditions.Environmental factors that may influence production can be temperature,pH, acidity, ethanol, sulfite, and availability of nutrients.

In addition to an appropriate carbon source, such as selected from oneof the herein disclosed types, bio-production media may contain suitableminerals, salts, cofactors, buffers and other components, known to thoseskilled in the art, suitable for the growth of the cultures andpromotion of the enzymatic pathway necessary for chemical productbio-production under the present disclosure. Another aspect of theinvention regards media and culture conditions that comprise geneticallymodified microorganisms of the invention and optionally supplements.

Typically cells are grown at a temperature in the range of about 25° C.to about 40° C. in an appropriate medium, as well as up to 70° C. forthermophilic microorganisms. Suitable growth media are wellcharacterized and known in the art.

Suitable pH ranges for the bio-production are between pH 2.0 to pH 10.0,where pH 6.0 to pH 8.0 is a typical pH range for the initial condition.However, the actual culture conditions for a particular embodiment arenot meant to be limited by these pH ranges.

Bio-productions may be performed under aerobic, microaerobic oranaerobic conditions with or without agitation.

In some cases, a change of an environmental condition comprises a changein sugar concentration of a culture medium contacting a cell. In somecases, a change in sugar concentration of a culture medium is anincrease of sugar concentration. In some other cases, a change in sugarconcentration is a decrease of sugar concentration. In some situations,an increase of sugar concentration is from 1% to 2%, from 2% to 3%, from3% to 4%, from 4% to 5%, from 5% to 10%, from 10% to 15%, from 15% to20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%,from 60% to 70%, from 70% to 80%, from 80% to 90%, or from 90% to 100%more sugar compared with the original sugar concentration in the culturemedium. In some situations, a decrease of sugar concentration is from 1%to 2%, from 2% to 3%, from 3% to 4%, from 4% to 5%, from 5% to 10%, from10% to 15%, from 15% to 20%, from 20% to 30%, from 30% to 40%, from 40%to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to90%, or from 90% to 100% less sugar compared with the original sugarconcentration in the culture medium.

In some cases, a change of an environmental condition comprises a changein oxygenation of a culture medium contacting a cell. In some cases, achange in oxygenation of a culture medium is an increase of oxygenation.In some other cases, a change in oxygenation of a culture medium is adecrease of oxygenation. In some situations, an increase of oxygenationis the addition of oxygen from 1% to 2%, from 2% to 3%, from 3% to 4%,from 4% to 5%, from 5% to 10%, from 10% to 15%, from 15% to 20%, from20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, from 60%to 70%, from 70% to 80%, from 80% to 90%, or from 90% to 100% more thanthe original amount of oxygen added in a culture medium. In somesituations, a decrease of oxygenation is the addition of oxygen from 1%to 2%, from 2% to 3%, from 3% to 4%, from 4% to 5%, from 5% to 10%, from10% to 15%, from 15% to 20%, from 20% to 30%, from 30% to 40%, from 40%to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to90%, or from 90% to 100% less than the original amount of oxygen addedin a culture medium.

Bio-production Reactors and Systems

Fermentation systems utilizing methods and/or compositions according tothe invention are also within the scope of the invention.

Any of the recombinant microorganisms as described and/or referred toherein may be introduced into an industrial bio-production system wherethe microorganisms convert a carbon source into a product in acommercially viable operation. The bio-production system includes theintroduction of such a recombinant microorganism into a bioreactorvessel, with a carbon source substrate and bio-production media suitablefor growing the recombinant microorganism, and maintaining thebio-production system within a suitable temperature range (and dissolvedoxygen concentration range if the reaction is aerobic or microaerobic)for a suitable time to obtain a desired conversion of a portion of thesubstrate molecules to a selected chemical product. Bio-productions maybe performed under aerobic, microaerobic, or anaerobic conditions, withor without agitation. Industrial bio-production systems and theiroperation are w-ell-known to those skilled in the arts of chemicalengineering and bioprocess engineering. The amount of a product producedin a bio-production media generally can be determined using a number ofmethods known in the art, for example, high performance liquidchromatography (HPLC), gas chromatography (GC), or GC/Mass Spectroscopy(MS).

Genic Modifications, Nucleotide Sequences, and Amino Acid Sequences

Embodiments of the present disclosure may result from introduction of anexpression vector into a host microorganism, wherein the expressionvector contains a nucleic acid sequence coding for an enzyme that is, oris not, normally found in a host microorganism.

The ability to genetically modify a host cell is essential for theproduction of any genetically modified (recombinant) microorganism. Themode of gene transfer technology may be by electroporation, conjugation,transduction, or natural transformation. A broad range of hostconjugative plasmids and drug resistance markers are available. Thecloning vectors are tailored to the host organisms based on the natureof antibiotic resistance markers that can function in that host. Also,as disclosed herein, a genetically modified (recombinant) microorganismmay comprise modifications other than via plasmid introduction,including modifications to its genomic DNA.

More generally, nucleic acid constructs can be prepared comprising anisolated polynucleotide encoding a polypeptide having enzyme activityoperably linked to one or more (several) control sequences that directthe expression of the coding sequence in a microorganism, such as E.coli, under conditions compatible with the control sequences. Theisolated polynucleotide may be manipulated to provide for expression ofthe polypeptide. Manipulation of the polynucleotide's sequence prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesequences utilizing recombinant DNA methods are well established in theart.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present disclosure. Thepromoter sequence may contain transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell. The techniques formodifying and utilizing recombinant DNA promoter sequences are wellestablished in the art.

For various embodiments of the invention the genetic manipulations maybe described to include various genetic manipulations, including thosedirected to change regulation of, and therefore ultimate activity of, anenzyme or enzymatic activity of an enzyme identified in any of therespective pathways. Such genetic modifications may be directed totranscriptional, translational, and post-translational modificationsthat result in a change of enzyme activity and/or selectivity underselected and/or identified culture conditions and/or to provision ofadditional nucleic acid sequences such as to increase copy number and/ormutants of an enzyme related to product production. Specificmethodologies and approaches to achieve such genetic modification arewell known to one skilled in the art.

In various embodiments, to function more efficiently, a microorganismmay comprise one or more gene deletions. For example, in E. coli, thegenes encoding the lactate dehydrogenase (ldhA), phosphateacetyltransferase (pta), pyruvate oxidase (poxB), pyruvateformate lyase(pflB), methylglyoxal synthase (mgsA), acetate kinase (ackA), alcoholdehydrogenase (adhE), the clpXP protease specificity enhancing factor(sspB), the ATPdependent Lon protease (Ion), the outer membrane protease(ompT), the arcA transcriptional dual regulator (arcA), and the iclRtranscriptional regulator (iclR) may be disrupted, including deleted.Such gene disruptions, including deletions, are not meant to belimiting, and may be implemented in various combinations in variousembodiments. Gene deletions may be accomplished by numerous strategieswell known in the art, as are methods to incorporate foreign DNA into ahost chromosome.

In various embodiments, to function more efficiently, a microorganismmay comprise one or more synthetic metabolic valves, composed of enzymestargeted for controlled proteolysis, expression silencing or acombination of both controlled proteolysis and expression silencing. Insome embodiments, a microorganism may comprise two, three, four, five,six, seven, eight, nine, or ten, or more synthetic metabolic valves. Forexample, one enzyme encoded by one gene or a combination of numerousenzymes encoded by numerous genes in E. coli may be designed assynthetic metabolic valves to alter metabolism and improve productformation. Representative genes in E. coli may include but are notlimited to the following: fabI, zwf gltA, ppc, udhA, lpd, sucD, aceA,pfkA, lon, rpoS, tktA or tktB. It is appreciated that it is well knownto one skilled in the art how to identify homologues of these genes andor other genes in additional microbial species.

For all nucleic acid and amino acid sequences provided herein, it isappreciated that conservatively modified variants of these sequences areincluded, and are within the scope of the invention in its variousembodiments. Functionally equivalent nucleic acid and amino acidsequences (functional variants), which may include conservativelymodified variants as well as more extensively varied sequences, whichare well within the skill of the person of ordinary skill in the art,and microorganisms comprising these, also are within the scope ofvarious embodiments of the invention, as are methods and systemscomprising such sequences and/or microorganisms.

Accordingly, as described in various sections above, some compositions,methods and systems of the present disclosure comprise providing agenetically modified microorganism that comprises both a productionpathway to make a desired product from a central intermediate incombination with synthetic metabolic valves to redistribute flux.

Aspects of the invention also regard provision of multiple geneticmodifications to improve microorganism overall effectiveness inconverting a selected carbon source into a selected product. Particularcombinations are shown, such as in the Examples, to increase specificproductivity, volumetric productivity, titer and yield substantiallyover more basic combinations of genetic modifications. In addition tothe above-described genetic modifications, in various embodimentsgenetic modifications, including synthetic metabolic valves also areprovided to increase the pool and availability of the cofactor NADPHand/or NADH which may be consumed in the production of a product.

More generally, and depending on the particular metabolic pathways of amicroorganism selected for genetic modification, any subgroup of geneticmodifications may be made to decrease cellular production offermentation product(s) other than the desired fermentation product,selected from the group consisting of acetate, acetoin, acetone,acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol,ethylene glycol, ethylene, propylene, butylene, isobutylene, ethylacetate, vinyl acetate, other acetates, 1,4-butanediol, 2,3-butanediol,butanol, isobutanol, sec-butanol, butyrate, isobutyrate,2-OH-isobutyrate, 3-OHbutyrate, ethanol, isopropanol, D-lactate,L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate,caprolactam, adipic acid, propanol, isopropanol, fusel alcohols, and1,2-propanediol, 1,3-propanediol, formate, fumaric acid, propionic acid,succinic acid, valeric acid, maleic acid and poly-hydroxybutyrate. Genedeletions may be made as disclosed generally herein, and otherapproaches may also be used to achieve a desired decreased cellularproduction of selected fermentation products other than the desiredproducts.

VI.A Gene Silencing

In particular the invention describes the use of controlled genesilencing to help enable the control over metabolic fluxes in controlledmulti-stage fermentation processes. There are several methodologiesknown in the art for controlled gene silencing, including but notlimited to mRNA silencing or RNA interference, silencing viatranscriptional repressors and CRISPR interference.

In some cases, a valve polynucleotide comprises a polynucleotideselected from the group consisting of: a silencing polynucleotide forrepressing transcription of a gene encoding said valve enzyme; adegradation polynucleotide for mediating cellular degradation of saidvalve enzyme; and a combination thereof.

In some cases, a valve polynucleotide comprises a silencingpolynucleotide, and said silencing polynucleotide comprises a guide RNA(gRNA) comprising a gRNA sequence that recognizes a promoter of a geneencoding said valve enzyme.

In some cases, a valve polynucleotide further encodes a CRISPR enzyme,wherein said CRISPR enzyme specifically binds to said promoter sequencewhen bound to said gRNA. In some cases, a CRISPR enzyme is catalyticallyinactive.

In some cases, a valve polynucleotide comprises a degradationpolynucleotide, wherein said degradation polynucleotide comprises asequence encoding a degradation tag, wherein said degradation tagmediates degradation of said valve enzyme. In some cases, the expressionof a valve polynucleotide is regulated by phosphate availability in acell. In some cases, the expression of a production polynucleotide isregulated by phosphate availability in a cell. In certain cases, thecell is an E. coli cell.

Controlled Proteolysis

In particular the current disclosure describes the use of controlledprotein degradation or proteolysis to help enable the control overmetabolic fluxes in controlled multi-stage fermentation processes. Thereare several methodologies known in the art for controlled proteindegradation, including but not limited to targeted protein cleavage by aspecific protease and controlled targeting of proteins for degradationby specific peptide tags. Systems for the use of the E. coli clpXPprotease for controlled protein degradation can be used. Thismethodology relies upon adding a specific C-terminal peptide tag such asa DAS4 (or DAS+4) tag. Proteins with this tag are not degraded by theclpXP protease until the specificity enhancing chaperone sspB isexpressed, sspB induces degradation of DAS4 tagged proteins by the clpXPprotease. In additional numerous site specific protease systems are wellknown in the art. Proteins can be engineered to contain a specifictarget site of a given protease and then cleaved after the controlledexpression of the protease. In some embodiments the cleavage can beexpected lead to protein inactivation or degradation. For example, anN-terminal sequence can be added to a protein of interest to enable clpSdependent clpAP degradation. In addition, this sequence can further bemasked by an additional N-terminal sequence, which can be controllablecleaved such as by a ULP hydrolase. This allows for controlled N-ruledegradation dependent on hydrolase expression. It is therefore possibleto tag proteins for controlled proteolysis either at the N-terminus orC-terminus.

The preference of using an N-terminal vs. C-terminal tag will largelydepend on whether either tag affects protein function prior to thecontrolled onset of degradation. The invention describes the use ofcontrolled protein degradation or proteolysis to help enable the controlover metabolic fluxes in controlled multi-stage fermentation processes,in E. coli. There are several methodologies known in the art forcontrolled protein degradation in other microbial hosts, including awide range of gram-negative as well as gram-positive bacteria, yeast andeven archaea. In particular, systems for controlled proteolysis can betransferred from a native microbial host and used in a non-native host.

Synthetic Metabolic Valve Control

In particular the current disclosure describes the use of syntheticmetabolic valves to control metabolic fluxes in multi-stage fermentationprocesses. There are numerous methodologies known in the art to induceexpression that can be used at the transition between stages inmultistage fermentations. These include but are not limited toartificial chemical inducers including: tetracycline,anhydrotetracycline, lactose, IPTG(isopropyl-beta-D-1-thiogalactopyranoside), arabinose, raffinose,tryptophan and numerous others. Systems linking the use of these wellknown inducers to the control of gene expression silencing and/orcontrolled proteolysis can be integrated into genetically modifiedmicrobial systems to control the transition between growth andproduction phases in multi-stage fermentation processes.

In addition, it may be desirable to control the transition betweengrowth and production in multi-stage fermentations by the depletion ofone or more limiting nutrients that are consumed during growth. Limitingnutrients can include but are not limited to: phosphate, nitrogen,sulfur and magnesium. Natural gene expression systems that respond tothese nutrient limitations can be used to operably link the control ofgene expression silencing and/or controlled proteolysis to thetransition between growth and production phases in multi-stagefermentation processes.

Products

In some embodiments, provided herein is a microorganism or a cell forproducing a product. In some cases, the product comprises3-hydroxypropionic acid. In some cases, the product comprises an aminoacid. In some cases, the amino acid comprises alanine. In some cases,the alanine is L-alanine. In some cases, the alanine is D-alanine. Insome cases, a rate of production of alanine is at least 0.1 g/L/hr, 0.2g/L/hr, 0.3 g/L/hr, 0.4 g/L/hr, 0.5 g/L/hr, 0.6 g/L/hr, 0.7 g/L/hr, 0.8g/L/hr, 0.9 g/L/hr, 1.0 g/L/hr, 1.1 g/L/hr, 1.2 g/L/hr, 1.3 g/L/hr, 1.4g/L/hr, 1.5 g/L/hr, 1.6 g/L/hr, 1.7 g/L/hr, 1.8 g/L/hr, 1.9 g/L/hr, 2.0g/L/hr, 2.5 g/L/hr, 3.0 g/L/hr, 3.5 g/L/hr, 4.0 g/L/hr, 4.5 g/L/hr, 5.0g/L/hr, 5.5 g/L/hr, 6.0 g/L/hr, 7.0 g/L/hr, 8.0 g/L/hr, 9.0 g/L/hr, orat least 10 g/L/hr.

In some cases, the alanine titers after 24 hours can be from 0 to 0.5g/L, 0.5 g/L to 1 g/L, 1 g/L to 1.5 g/L, 1.5 g/L to 2 g/L, 2 g/L to 2.5g/L, 2.5 g/L to 3 g/L, 3 g/L to 3.5 g/L, 3.5 g/L to 4 g/L, 4 g/L to 4.5g/L, 4.5 g/L to 5 g/L, or from 5 g/L to 10 g/L. The dynamic range ofalanine production offered by SMVs can be up to a 4-fold increasecompared to that offered by solely altering the expression level of theproduction pathway enzymes (by changing the promoter). In some cases,the dynamic range of alanine production offered by SMVs can be up to a2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or10-fold increase compared to that offered by solely altering theexpression level of the production pathway enzymes.

In some cases, a production polynucleotide in the microorganism encodesan alanine exporter. In some cases, the alanine exporter is alaE.

In some cases, the product comprises mevalonic acid. In some cases, arate of production of mevalonic acid is at least 0.1 g/L/hr, 0.2 g/L/hr,0.3 g/L/hr, 0.4 g/L/hr, 0.5 g/L/hr, 0.6 g/L/hr, 0.7 g/L/hr, 0.8 g/L/hr,0.9 g/L/hr, 1.0 g/L/hr, 1.1 g/L/hr, 1.2 g/L/hr, 1.3 g/L/hr, 1.4 g/L/hr,1.5 g/L/hr, 1.6 g/L/hr, 1.7 g/L/hr, 1.8 g/L/hr, 1.9 g/L/hr, 2.0 g/L/hr,2.5 g/L/hr, 3.0 g/L/hr, 3.5 g/L/hr, 4.0 g/L/hr, 4.5 g/L/hr, 5.0 g/L/hr,5.5 g/L/hr, 6.0 g/L/hr, 7.0 g/L/hr, 8.0 g/L/hr, 9.0 g/L/hr, or at least10 g/L/hr.

Methods

Provided herein are methods for producing a product in an engineeredmicroorganism in a large scale. Also provided herein are methods forengineering microorganisms for large-scale production of a product in ahigh-throughput fashion.

In some cases, provided herein is a method, comprising: culturing aplurality of strains of a cell, wherein each strain of said plurality ofstrains comprises (i) an engineered valve polynucleotide for mediatingcontrolled reduction of expression of a valve enzyme acting in ametabolic pathway, wherein said controlled reduction of expression ofsaid valve enzyme reduces flux through said metabolic pathway; and (ii)an engineered production polynucleotide for mediating controlledincrease in expression of a production enzyme for generation of saidproduct; wherein each strain of said plurality of strains differs fromanother strain in a sequence of at least one of said engineered valvepolynucleotide or said engineered production polynucleotide; measuring alevel of said product generated by each of said plurality of strains;and selecting a strain based on said level of said product. In someembodiments, the method further comprises growing said selected strainin a bioreactor. In some embodiments, a culture medium comprising saidselected strain has a volume of at least 100 ml, 200 ml, 300 ml, 400 ml,500 ml, 60 ml, 700 ml, 800 ml, 900 ml, or at least 1000 ml. In someembodiments, a culture medium has a volume of at least 1 L.

In some embodiments, a valve polynucleotide comprises a polynucleotideselected from the group consisting of: a silencing polynucleotide forrepressing transcription of a gene encoding said valve enzyme; adegradation polynucleotide for mediating cellular degradation of saidvalve enzyme; and a combination thereof. In some embodiments, a firstand a second strain of said plurality of strains comprise a silencingpolynucleotide. In some embodiments, a silencing polynucleotidecomprises a guide RNA (gRNA) comprising a gRNA sequence that recognizesa promoter of a gene encoding said valve enzyme. In some embodiments, agRNA sequence differs between said first and second strains. In someembodiments, a promoter recognized by said gRNA differs between saidfirst and second strains. In some embodiments, a first strain comprisessaid silencing polynucleotide and said degradation polynucleotide, and asecond strain comprises said silencing polynucleotide but does notcomprise said degradation polynucleotide. In some embodiments, a levelof product is greater in said second strain than said first strain. Insome embodiments, a level of product is greater in said first strainthan said second strain. In some embodiments, a valve enzyme comprisesan enzyme selected from the group consisting of enoyl-ACP/CoA reductase(fabI), glucose-6-phosphate dehydrogenase (zwf), lipoamide dehydrogenase(lpd), citrate synthase (gltA), soluble transhydrogenase (udhA).NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapA), and acombination thereof. In some embodiments, a production enzyme comprisesan enzyme selected from the group consisting of NADPH-dependent alaninedehydrogenase (aid), alanine exporter (alaE), NADPH-dependentglyceraldehyde-3-phosphate dehydrogenase (gapN), and a combinationthereof.

In some embodiments, a product is selected from the group consisting ofmevalonic acid, 3-hydroxypropionic acid, an amino acid, and acombination thereof. In some embodiments, the amino acid is alanine. Insome embodiments, the alanine is L-alanine. In some embodiments, thealanine is D-alanine.

In some embodiments, a rate of production of the product during saidstationary phase is reduced less in response to a change of anenvironmental condition as compared to a cell lacking said controlledreduction of expression of said valve enzyme.

In some embodiments, a change of an environmental condition comprises achange in a sugar concentration of a culture medium contacting saidcell.

In some embodiments, a change of an environmental condition comprises achange in oxygenation of a culture medium contacting said cell.

In some cases, provided herein is a method of generating a cellularproduct comprising: culturing a heterologous cell in a culture medium,wherein said heterologous cell comprises: (i) an engineered valvepolynucleotide for mediating controlled reduction of expression of avalve enzyme acting in a metabolic pathway, wherein said controlledreduction of expression of said valve enzyme reduces flux through saidmetabolic pathway, wherein said controlled reduction of expression ofsaid valve enzyme induces a stationary phase of said cell; and (ii) anengineered production polynucleotide for mediating controlled increasein expression of a production enzyme for generation of said product;wherein a rate of production of said product during said stationaryphase is reduced less in response to a change of an environmentalcondition as compared to a cell lacking said controlled reduction ofexpression of said valve enzyme. In some embodiments, the method furthercomprises changing said environmental condition. In some embodiments,the environmental condition comprises a sugar concentration of saidculture medium, and changing said environmental condition comprisesincreasing or decreasing said sugar concentration. In some cases, saidsugar is glucose, sucrose, lactose, maltose, xylose, mannitol, or acombination thereof. In some cases, said sugar is glucose. In somecases, the environmental condition comprises an oxygen concentration ofsaid culture medium, and changing said environmental condition comprisesincreasing or decreasing said oxygen concentration. In some cases, saidculturing is performed in a bioreactor.

In some cases, said culture medium has a volume of at least 100 ml, 200ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, or at least1000. In some cases, said culture medium has a volume of at least 1 L.In some case, said product comprises 3-hydroxypropionic acid. In somecases, said product comprises an amino acid. In some cases, said aminoacid comprises alanine.

In some cases, a rate of production of said alanine is at least 0.1g/L/hr, 0.2 g/L/hr, 0.3 g/L/hr, 0.4 g/L/hr, 0.5 g/L/hr, 0.6 g/L/hr, 0.7g/L/hr, 0.8 g/L/hr, 0.9 g/L/hr, 1.0 g/L/hr, 1.1 g/L/hr, 1.2 g/L/hr, 1.3g/L/hr, 1.4 g/L/hr, 1.5 g/L/hr, 1.6 g/L/hr, 1.7 g/L/hr, 1.8 g/L/hr, 1.9g/L/hr, 2.0 g/L/hr, 2.5 g/L/hr, 3.0 g/L/hr, 3.5 g/L/hr, 4.0 g/L/hr, 4.5g/L/hr, 5.0 g/L/hr, 5.5 g/L/hr, 6.0 g/L/hr, 7.0 g/L/hr, 8.0 g/L/hr, 9.0g/L/hr, or at least 10 g/L/hr. In some cases, said productionpolynucleotide encodes an alanine exporter. In some cases, said alanineexporter is alaE. In some cases, said culturing occurs for less than 20hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90hours, or less than 100 hours. In some cases, said culturing occurs forless than 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40hours, or less than 45 hours.

In some cases, said culturing occurs for less than 30 hours.

In some cases, said product comprises mevalonic acid. In some cases, arate of production of said mevalonic acid is at least 0.1 g/L/hr, 0.2g/L/hr, 0.3 g/L/hr, 0.4 g/L/hr, 0.5 g/l/hr, 0.6 g/L/hr, 0.7 g/L/hr, 0.8g/L/hr, 0.9 g/L/hr, 1.0 g/L/hr, 1.1 g/L/hr, 1.2 g/L/hr, 1.3 g/L/hr, 1.4g/L/hr, 1.5 g/L/hr, 1.6 g/L/hr, 1.7 g/L/hr, 1.8 g/L/hr, 1.9 g/L/hr, 2.0g/L/hr, 2.5 g/L/hr, 3.0 g/L/hr, 3.5 g/L/hr, 4.0 g/L/hr, 4.5 g/L/hr, 5.0g/L/hr, 5.5 g/L/hr, 6.0 g/L/hr, 7.0 g/l/hr, 8.0 g/L/hr, 9.0 g/L/hr, orat least 10 g/L/hr. In some cases, said culturing occurs for less than20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90hours, or less than 100 hours. In some cases, said culturing occurs forless than 80 hours.

In some embodiments, a valve polynucleotide comprises a polynucleotideselected from the group consisting of: a silencing polynucleotide forrepressing transcription of a gene encoding said valve enzyme; adegradation polynucleotide for mediating cellular degradation of saidvalve enzyme, and a combination thereof. In some cases, a valvepolynucleotide comprises a silencing polynucleotide, and said silencingpolynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequencethat recognizes a promoter of a gene encoding said valve enzyme. In somecases, a valve polynucleotide further encodes a CRISPR enzyme, whereinsaid CRISPR enzyme specifically binds to said promoter sequence whenbound to said gRNA. In some cases, a CRISPR enzyme is catalyticallyinactive. In some case, a valve polynucleotide comprises a degradationpolynucleotide, wherein said degradation polynucleotide comprises asequence encoding a degradation tag, wherein said degradation tagmediates degradation of said valve enzyme. In some cases, an expressionof said valve polynucleotide is regulated by phosphate. In some cases,an expression of said production polynucleotide is regulated byphosphate. In some cases, said cell is an E. coli cell.

Optimization of Bio-Production

Biotechnology based fermentation processes have been successfullydeveloped to produce everything from biologics and small moleculetherapeutics to specialty, bulk and commodity chemicals, and even nextgeneration biofuels¹⁻³. These processes have made rapid advancements inrecent years due to numerous technology developments^(4, 5). It hasnever been easier to produce new molecules using synthetic biology.Despite these advances, a major challenge remains in taking moleculesfrom proof of concept (POC) to commercially meaningful levels. Strainoptimization, or overcoming the “mg” to “kg” hurdle has remained a keybarrier to the successful commercialization of bio-processes. After thedemonstration of POC, successful bio-process development routinelyrequires lengthy iterations of both microbial strain and fermentationoptimization⁶⁻⁸ (FIG. 1B). These optimization efforts are often specificto the product or host strain of interest. The throughput of syntheticbiology has outpaced that of metabolic engineering, partly due to a lackof broadly useful tools to perform meaningful and standardizedoptimization of engineered microbial strains in a high-throughputmanner⁹.

There are numerous challenges in strain optimization and moving past POClevels, not the least of which are the size and complexity of thepotential design space. In contrast to simpler gene circuits, amenableto electrical circuit models¹⁰⁻¹², metabolic networks are highlyinterconnected. Each metabolite and/or enzyme can interact with endlessothers. This combinatorial complexity results in a huge potential designspace, which is intractable to the kinds of systematic experimentationrequired for the development of standardized design principles(Supplemental Materials, Table 1). The challenges in addressing such alarge design space have persisted despite the dramatic advances in, anddecreased costs of, reading and writing DNA that have led to newhigh-throughput DNA assembly and microbial strain constructionmethods¹³⁻¹⁶. It is not surprising that new synthetic biologytechnologies involving strain engineering are often demonstrated witheasily screened or selected phenotypes^(13, 17-19). Most of these arelimited to a focus on optimizing a limited set of pathway specificenzymes.

One approach to overcome the complexity of this challenge is the use ofin vitro systems for bio-production, which comprise a limited set ofmetabolic enzymes. However, these approaches have challenges inreplicating key advantages of in vivo systems, including cofactorrecycling and energy generation^(20, 21). Another approach to deal withthis complexity is to develop faster screening methods for strainevaluation²². However, increased throughput alone can never evaluate thefull complexity of the potential design space. In addition, resultsobtained from high-throughput studies often do not translate, even inthe same microbe, to a different environment^(20, 23, 24). Small scalescreens do not readily translate to larger scale production processes,leading to iterations of process optimization on top of strainoptimization (FIG. 1B). This is because metabolism is highly regulatedand can respond, sometimes dramatically, to changes in environmentalconditions^(25 20, 26-28). A lack of environmental robustness istraditionally one factor making the scale up of fermentation basedprocesses difficult. This issue has led to the development ofspecialized complex micro-reactor systems for scale down offering onlymodest improvements in throughput^(20, 29-31).

There remains a significant need for broadly applicable, rapid androbust approaches to greatly reduce the time and costs transitioningfrom “mgs” to “kgs”. Ideally, approaches should be amenable to multipleproducts and production hosts. Provided herein is the development of ageneralizable, high-throughput strain optimization approach that enablesthe use of truly scalable, standardized fermentation processes. Thisapproach, as outlined in FIG. 1B, panel b, involves the dynamicminimization of the active metabolic network³², which combines thebenefits of a smaller design space common to in vitro approaches whilemaintaining the benefits of in vivo biosynthesis²⁰. We can isolate andfocus on the minimal metabolic networks required for production.Utilizing combinations of synthetic metabolic valves (SMVs)^(32, 33)(FIGS. 2A-D) we can dynamically minimize the metabolic network andredirect metabolic flux in the context of a standardized 2-stagefermentation process²⁰.

This approach can reduce the complexity of the problem and the size ofthe relevant design space, greatly speeding up optimization. In variousembodiments, it is demonstrated herein that dynamic metabolic networkminimization can improve pathway fluxes beyond those achievable withproduction pathway modifications alone (FIGS. 3A-K and 6A-H).

Simultaneously, we demonstrate that dynamic network minimization reducesmetabolic responses to environmental conditions, which increases therobustness and scalability of engineered strains (FIGS. 3A-K and SA-J).

Examples

2-Stage Synthetic Metabolic Valves is E. coli

We first developed improved synthetic metabolic valves (SMVs) in E. colithat are capable of the dynamic reduction of protein levels in a 2-stageprocess. These SMVs can be used to reduce levels of key metabolicenzymes (or reduce enzymatic activities of key metabolic enzymes) andrely on controlled proteolysis or CRISPR-based gene silencing or bothproteolysis and silencing in combination (FIGS. 2A-D)³²⁻³⁵. Cell growthand dynamic metabolic control can be implemented using phosphatedepletion as an environmental trigger. Phosphate can be an idealcandidate as a trigger, as one of the costliest components of minimalmedia. In addition, stationary phases induced in E. coli by phosphatedepletion have retained glycolytic uptake as well as increased proteinexpression^(31, 36). Numerous promoter systems responding to phosphateare well characterized in E. coli as well as other microbes including S.cerevisiae ³⁷. Phosphate responsive promoter variants were evaluated(Supplemental Materials, Section 1) and subsequently used for 2-stagecontrol.

SMVs were implemented in E. coli using the native Type I-E CascadeCRISPR system for induced gene silencing^(34,38), while controlledproteolysis was induced by incorporating C-terminal degron tags ontarget proteins, both as previously demonstrated^(63,33) (FIG. 2A).These systems were introduced into a host strain initially engineeredfor minimal byproduct formation and high biomass yields and growth rates(E. coli strain DLF_0025, Supplemental Materials, Section3)^(24, 27, 28, 39). Using this approach, as FIGS. 2A-D demonstrate,protein levels can be controlled in 2-stage processes, as exemplified byturning “ON” GFPuv and “OFF” mCherry fluorescent proteins with phosphatedepletion in minimal medium. The combination of gene silencing withproteolysis results in the largest rates of protein degradation (FIGS.2C-D). The specific impact of gene silencing and proteolysis on decayrates will likely vary depending on the host, target gene/enzyme, andits specific natural turnover rates and expression levels.

Metabolic Network Minimization Leads to Improved Fluxes

With the successful demonstration of dynamic control of protein levelsin a 2-stage process, we turned to investigate the dynamic control ofmetabolic fluxes in E. coli through controlled reduction of key centralmetabolic enzymes alone and in combination. Reducing fluxes throughthermodynamically favored “committed” reactions in the network isexpected to lead to increases in network metabolite pools (SupplementalMaterials Section 5), and as a result, changes in pathway fluxes.Enzymes in key committed steps in central metabolic pathways wereidentified and chosen as initial SMV targets and alanine was chosen asan initial test product (FIGS. 3A-K). A set of strains were constructedfor alanine production (FIG. 3A), comprising an NADPH-dependent alaninedehydrogenase (ald*)⁴². Variants with multiple combinations of SMVs incentral metabolic enzymes were made, with either modifications to induceproteolysis or gene silencing or both in combination. (SupplementalMaterials, Section 3). Together the set of strains having SMVs evaluatedin 2-stage processes are identified as “Valve” strains. A panel ofalanine “Valve” strains (˜500 strains in total) were evaluated foralanine production in standardized, 2-stage, 96-well plate basedmicro-fermentations (Supplemental Materials, Section 7). Alanine titersafter 24 hours of production are given in FIGS. 3B-C. Briefly, alaninetiters after 24 hours ranged from ˜0 g/L to ˜4.7 g/L, and as expected,varied significantly with respect to the number and combination of SMVs;most SMV combinations lead to improved performance when compared to thecontrol with no SMVs and the alanine pathway alone. In some cases, thealanine titers after 24 hours can be from 0 to 0.5 g/L, 0.5 g/L to 1g/L, 1 g/L to 1.5 g/L, 1.5 g/L to 2 g/L, 2 g/L to 2.5 g/L, 2.5 g/L to 3g/L, 3 g/L to 3.5 g/L, 3.5 g/L to 4 g/L, 4 g/L to 4.5 g/L, 4.5 g/L to 5g/L, or from 5 g/L to 10 g/L. The dynamic range of alanine productionoffered by SMVs can be up to a 4-fold increase compared to that offeredby solely altering the expression level of the production pathwayenzymes (by changing the promoter) (Supplemental Materials. Section 7).In some cases, the dynamic range of alanine production offered by SMVscan be up to a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, or 10-fold increase compared to that offered by solely alteringthe expression level of the production pathway enzymes. Importantly, theuse of proteolysis or silencing alone and/or in combination hadsignificant impacts on production, indicating that for each enzyme thefine tuning of activity using SMVs is critical. One of the bestperforming strains from the micro-fermentations was then evaluated in aminimal medium, 2-stage, 1 L fermentation with 10 gdcw/L of biomass(FIG. 3F), which resulted in 80 g/L 100% L-alanine after 48 hours ofproduction with a yield of 0.8 gig. Further engineering of this strainby overexpressing an alanine exporter (encoded by the E. coli alaEgene⁴³) resulted in 147 g/L 100% L-alanine after 27 hours of productionwith a yield within error of theoretical yield ˜1 g/g, (FIG. 3G).

Micro-fermentation Robustness

A central hypothesis was that by restricting metabolism in theproduction stage, strain performance could not only be improved, butwould be more robust to environmental (process) conditions. Simply put,carbon flow is restricted through a minimized metabolic network, whichcan no longer adapt via cellular responses to the environment. To testthis hypothesis, strains were evaluated under different“micro-fermentation” process conditions. Glucose concentration andoxygen transfer rate (key process variables impacting strain performancein traditional fermentations²⁶) were varied (FIG. 3D, SupplementalMaterials, Section 8), and alanine production measured. A robustnessscore (RS) was developed to quantify environmental robustness. Larger RSscores indicate more robust strains. Whereas relative standard deviation(RSD) is one metric for robustness, we wanted to incorporate a strictermeasure of robustness which also incorporates the maximal deviation (MaxDev) a strain has under all process conditions (RS, Equation (1)).

$\begin{matrix}{{RS} = {100 - {\frac{{{average}({RSD})} + {\max({Dev})}}{2}*100}}} & {{Equation}\mspace{14mu}(1)}\end{matrix}$

Robustness scores for a subset of 48 alanine “Valve” strains are givenin FIG. 3E. Results from these experiments studies are tabulated inSupplemental Materials, Section 8. A Chi² analysis using a cutoff ofRS>0.6 for robustness was used to identify key SMVs which statisticallycontribute to process robustness. The proteolytic degradation of fabIwas a primary contributor to robustness (Chi²=13.85, P_(value)<0.001)and as a result. “Valve” strains with proteolytic degradation of fabIwere used in further studies. In addition, the “Valve” strains withproteolytic degradation of gltA and/or the combination of theproteolytic degradation of fabI and gltA were found to also besignificant contributors of robustness, albeit with a large P_(value).

2-Stage “Value” Strains Compared to Traditional Growth AssociatedStrains

To compare the 2-stage approach enabled by SMVs to more traditionalgrowth associated processes, we constructed 5 strains, withconstitutively expressed alanine dehydrogenase (ald*), capable of thegrowth associated production of alanine. These growth associated strainsvaried in the strength of the promoter used to drive ald* expression⁴⁴(Supplemental Materials, Section 2), yet utilized the same commonno-valve control host strain. FIG. 5 illustrates the results of a directcomparison of “Valve” strains in a 2-stage process compared to “GrowthAssociated (GA)” strains in a traditional fermentation at the microtiter(FIGS. 5A-D) and 1 L (FIGS. 5E-J) scales. In micro-fermentations,2-stage “Valve” strains outperformed GA strains with respect to titerand process robustness. The most robust GA strain from themicro-fermentation analysis (also with the highest production level) wascompared to a robust “Valve” strain in 1 L fermentations with variedprocess conditions. The “Valve” strains showed consistent performance inall process conditions evaluated (FIG. 5E), consistent with results frommicro-fermentations, where the GA strain had significant performancevariability dependent on process. We hypothesized that the increasedenvironmental robustness observed in both “micro-” and 1 L scalefermentations for “Valve” strains would lead to predictable scale up,where strains with improved performance in high-throughputmicro-fermentations would reliably have improved performance incontrolled bioreactors. To evaluate the scalability of the system.“Valve” alanine strains with statistically differentiated performance inmicro-fermentations (P-value<0.001) were evaluated in standardized2-stage 1 L fermentations and compared to all GA strains. Statisticallydifferent performances observed in “micro-fermentations” have scaledpredictably to 1 L fermentations for 2-stage “Valve” strains. Thiscontrasts with results obtained with GA strains where no correlationbetween micro-fermentation and 1 L performance was observed (FIGS.5G-H).

Product Flexibility

With the successful and predictable scale-up of alanine strains into 1 Lfully instrumented fermentations, we moved to validate the technologyplatform for an additional product: mevalonic acid. To this end,additional dynamic production pathways were constructed for mevalonicacid biosynthesis (FIG. 6A). A set of two-gene production pathwayplasmids encoding three enzymatic functions was constructed formevalonic acid production, consisting of the E. faecalis mvaE and mvaSgenes encoding a bifunctional acetyl-CoA acetyltransferase, NADPHdependent HMG-CoA reductase, and HMG-CoA synthase respectively. A mutantmvaS gene, mvaS(A1110G) with higher activity was used^(45, 46).Production plasmids were initially evaluated for mevalonate productionin the control strain (FIG. 6B). The best producing plasmid was thenintroduced into a variety of engineered “Valve” strains and evaluated inmicro-fermentations (FIG. 6C). A subset of statistically differentiatedstrains were then evaluated in 1 L fermentations to assess scalability(FIG. 6D), which, as in the case of alanine, was predictive. In somecases, a performing strain produced meaningful titers and yields, 97 g/Lin 78 hrs of production with a yield of 0.46 g/g (84% of theoreticalyield) (FIG. 6E). Specific productivity for this mevalonate strain isover 4-fold higher than the best previously reported results⁴⁷(Supplemental Materials, Section 9).

Discussion

Historically some of the most successful efforts to metabolicallyengineer the production of small molecules have leveraged the power ofanaerobic metabolism to couple product formation with growth. This hasallowed for the classical design and selection of industrial strains toproduce many products including ethanol, succinic acid, lactate andisobutanol, which have leveraged the power of evolution and selection toreach optimal metabolic fluxes in engineered networks^(48, 49)_ENREF_2.While growth associated production is not strictly linked to anaerobicmetabolism, growth association greatly limits the number and variety ofdifferent molecules that can be made using synthetic biology. A generic,robust and accessible non-growth associated platform would greatlysimplify the optimization and scale up of a diverse number of products.

In contrast to most existing 2-stage processes, which have relied onnatural metabolic responses to environmental triggers for productionimprovement, we have taken the next step in actively minimizing theessential metabolic network and redirecting metabolites to products ofinterest. Many of the targeted essential central metabolic pathways inthis work have traditionally been off limits to engineering strategies,as deleting essential enzymes is incompatible with growth and growthassociated production in traditional fermentation. The dynamicallyminimized metabolic network also results in enhanced robustness toenvironmental variables enabling the faithful translation ofhigh-throughput small-scale studies to larger instrumentedfermentations. A current paradigm in the field is to improve thethroughput of relevant strain evaluations by developing small-scale,custom-designed micro-reactors for enhanced process control. Incontrast, our approach is a move in a new direction involvingengineering microbial metabolism to be less sensitive to processchanges, simplifying high-throughput experimentation.

Beyond robustness, we have demonstrated that combinatorial modificationsto essential enzymes in minimal metabolic networks can lead tosignificant improvements in production, particularly when compared toaltering production pathway expression levels alone. These largevariations in performance are due to changes in a limited subset of keycentral metabolic nodes, likely resulting in altered metabolite levels.Compared to previous approaches to dynamically control enzyme levels, wedemonstrate improved potential for fine tuning of protein levels with acombination of gene silencing and proteolysis⁵⁰. As stationary phasecells cannot dilute existing proteins with cell division, this dualapproach makes sense. The specific control of the level of any givenenzyme will of course also depend on natural turnover mechanisms. Atfirst glance, it may still be surprising that the combination of bothgene silencing and proteolysis together does not always result inimproved performance. i.e. “more is not always better”. Future effortsmay be needed to explain these results, which could either be due to arequirement of maintaining minimal fluxes in the larger network or aconsequence of changes in the levels of key regulatory metabolites thatare not part of the minimal network, yet influence network activity.

While the approach as demonstrated can address many issues common tomost bio-production processes, many product specific challenges remain.The toxicity of a product or pathway metabolite may limit titers orproduction rates. A minimal network that may be optimal at a low titer,may not be optimal at elevated titers. In addition, the engineering ofimproved enzymes is often a challenge in many “mg” to “kg” projects.

Feasibility of adapting this approach to other microbial hosts isexpected. Key requirements for new hosts include a rapid and robustgrowth phase, the ability to engineer dynamic control over proteinlevels, and a metabolically active stationary phase. Numerous microbeshave well characterized nutrient triggers for productive stationaryphase metabolism³⁶, for example nitrogen limitation in Ralstoniaspecies, Yarrowia species and others⁵¹ ⁵². Even when these requirementsare not naturally met, they can be engineered into the host such as S.cerevisiae or other microbes, with each potential host presenting uniquechallenges and corresponding solutions.

Future efforts can be aimed at applying this platform for molecules withmore complex production pathways. This approach can offer a tractableroute for rapid optimization to metabolic engineers and syntheticbiologists, who wish to move past POC levels and begin to tackleproblems at more industrially relevant rates, titers and yields.

Methods Reagents and Media

Unless otherwise stated, all materials and reagents were of the highestgrade possible and purchased from Sigma (St. Louis, Mo.). C13 labeledAlanine (2,3-13C2, 99%) (Item #CLM-2734-PK) was purchased from CambridgeIsotope Laboratories. Inc. (Tewksbury, Mass.). Luria Broth was used forroutine strain and plasmid propagation and construction. Workingantibiotic concentrations were as follows: ampicillin (100 μg/mL),kanamycin (35 μg/mL), chloramphenicol (35 μg/mL), spectinomycin (100μg/mL), zeocin (50 μg/mL), gentamicin (10 μg/mL), blasticidin (100μg/mL), puromycin (150 μg/mL), tetracycline (5 μg/mL). Luria broth withlow salt (Lennox formulation) was used to select for zeocin, blasticidinand puromycin resistant clones. In addition, for puromycin selection,phosphate buffer (pH=8.0) was added to LB Lennox to a finalconcentration of 50 mM. Media formulations including stock solutions aredescribed in Supplemental Materials. Section 7.

E. coli Strain Construction

Oligonucleotides and synthetic linear DNA (Gblocks™) used for strainconstruction and confirmation are all given in Supplemental Materials,Section 3, and they were obtained from Integrated DNA Technologies (IDT,Coralville, Iowa). Strain BW25113 was obtained from the Yale GeneticStock Center (CGSC http://cgsc.biology.yale.edu/). Strain BWapldf was akind gift from George Chen (Tsinghua University)⁶². Chromosomalmodifications were made using standard recombineering methodologies⁶³either with direct antibiotic cassette integration in the case ofC-terminal DAS+4 tags carrying antibiotic resistance cassettes, orthrough scarless tet-sacB selection and counterselection, strictlyfollowing the protocols of Li et al⁶⁴. The recombineering plasmid pSIM5and the tet-sacB selection/counterselection marker cassette were kindgifts from Donald Court (NCI,https://redrecombineering.ncifcrf.gov/court-lab.html). Briefly, thetet-sacB selection/counterselection cassette was amplified using theappropriate oligos supplying ˜50 bp flanking homology sequences usingEconotaq (Lucigen Middleton, Wis.) according to manufacturer'sinstructions, with an initial 10 minutes denaturation at 94° C.,followed by 35 cycles of 94° C., for 15 seconds, 52° C. for 15 seconds,and 72° C. for 5 minutes. Cassettes used for “curing” of the tet-sacBcassette or direct integration (when an antibiotic marker is present)were obtained as gBlocks from IDT. In the case of the sspB genedeletion, the open reading frame deletion replaced with a kanamycinresistance was amplified from the Keio Collection strain, JW3197-1⁶⁵,and moved to the appropriate background strain using standardmethodologies. The kanamycin resistance cassette was cured using thepCP20 plasmid, leaving an frt scar^(63,65). Chromosomal modificationswere confirmed by PCR amplification and sequencing (Eton Biosciences)using paired oligonucleotides, either flanking the entire region, or inthe case of DAS+4 tag insertions an oligo 5′ of the insertion and oneinternal to the resistance cassette.

E. coli Plasmid Construction

Primers used for the design and construction of CASCADE guides arrayswere listed in Supplemental Materials, Section 6. Gene silencing guidearrays were expressed from a series of pCASCADE plasmids. ThepCASCADE-control plasmid was prepared by swapping the pTet promoter inpcrRNA.Tet⁷³ with an insulated low phosphate induced ugpB promoter⁷⁴.Promoter sequences for all genes were obtained from EcoCyc database(https://ecocyc.org/). In order to design CASCADE guide array. CASCADEPAM sites near the −35 or −10 box of the promoter of interest wereidentified, 30 bp at the 3′ end of PAM site was selected as the guidesequence and cloned into pCASCADE plasmid using Q5 site-directedmutagenesis (NEB, MA) following manufacturer's protocol, with themodification that 5% v/v DMSO was added to the Q5 PCR reaction. PCRcycles were as follows: amplification involved an initial denaturationstep at 98° C. for 30 second followed by cycling at 98° C. for 10second, 72° C. for 30 second, and 72° C. for 1.5 min (the extension ratewas 30 second/kb) for 25 cycles, then a final extension for 2 min at 72°C. 2 μL of PCR mixture was used for 10 μL KLD reaction, which proceededunder room temperature for 1 hour, after which, 1 μL KLD mixture wasused for electroporation.

The pCASCADE guide array plasmids were prepared by sequentiallyamplifying complementary halves of each smaller guide plasmid by PCR,followed by subsequent DNA assembly. The pCASCADE-control vector wasused as template, pCASCADE plasmids with arrays of two or more guideswere prepared using Q5 High-Fidelity 2× Master Mix (NEB, MA). PCR cycleswere as follows: amplification involved an initial denaturation step at98° C. for 30 second followed by cycling at 98° C. for 10 second, 66° C.for 30 second, and 72° C. for 45 second (the extension rate was 30second/kb) for 35 cycles, then a final extension for 2 min at 72° C. PCRproduct was purified by gel-extraction, 20 μL ultrapure water was usedto elute 50 μL PCR reaction purification, 1 μL of each eluted PCRproduct was used for 10 μL of Gibson Assembly (NEB, MA), which wascompleted by incubation at 50° C. for 15 min. 1 μL Gibson Assembly mixwas used for electroporation.

Production pathways enzymes were expressed from high copy plasmids vialow phosphate inducible promoters. Production pathway gene sequenceswere codon optimized using the Codon Optimization Tool from the IDTwebsite, phosphorylated G-blocks™ were designed and purchased from IDTfor each pathway. Plasmids were assembled using NEBuilder® HiFi DNAAssembly Master Mix following manufacturer's protocol (NEB, MA),pSMART-HC-Kan (Lucigen, WI) was used as backbone for all pathwayplasmids. All plasmid sequences were confirmed by DNA sequencing (EtonBioscience, NC) and deposited with Addgene.

E. coli BioLector

Single colonies of each strain were inoculated into 5 mL LB withappropriate antibiotics and cultured at 37° C. 220 rpm for 9 hours oruntil OD600 reached >2. 500 μL of the culture was inoculated into 10 mLSM10 medium with appropriate antibiotics, and cultured in a square shakeflask (CAT#: 25-212, Genesee Scientific, Inc. San Diego, Calif.) at 37°C., 220 rpm for 16 hours. Cells were pelleted by centrifugation and theculture density was normalized to OD600 5 using FGM3 media. Growth andfluorescence measurements were obtained in a Biolector (m2p labs,Baesweiler, Germany) using a high mass transfer FlowerPlate (CAT#:MTP-48-B, m2p-labs, Germany), 40 μL of the OD normalized culture wasinoculated into 760 μL of FGM3 medium with appropriate antibiotics.Biolector settings were as follows: RFP gain=100, GFP gain=20, Biomassgain=20, shaking speed=1300 rpm, temperature=37° C. humidity=85%. Everystrain was analyzed in triplicate.

E. coli Micro-Fermentations

Plasmids were transformed into host strains by electroporation using ECM630 High Throughput Electroporation System (Harvard Apparatus. Inc.Holliston, Mass.) following manufacturer's protocol or using individualelectroporation cuvettes. Glycerol stocks were prepared for eachtransformation plate by adding equal volume of sterile 20% glycerol, and3 μL were used to inoculate overnight culture in 150 μL SM10++ mediumwith appropriate antibiotics. Plates were covered with sandwich covers(Model #CR15% obtained from EnzyScreen, Haarlam, The Netherlands). Thesecovers ensured minimal evaporative loss during incubation. Unlessotherwise stated, 96 well plates were cultured at 37° C., 404 rpm for 16hours, shaker orbit is 25 mm. This combination of orbit and minimalshaking speed is required to obtain needed mass transfer coefficient andenable adequate culture oxygenation.

After 16 hours of growth, cells were pelleted by centrifugation, excessmedia was removed and cells were resuspended in 150 μL of FGM3 Washsolution. Subsequently cells were once again pelleted and again excessmedia was removed, pellet was resuspended in 50 μL FGM3 No Phosphatemedia containing appropriate antibiotics, 5 μL of the resuspendedculture was added to 195 μL of water for OD600 measurement usingstandard flat bottom 96 well plate. OD600 for production was normalizedto OD600=1, using FGM3 No Phosphate media containing appropriateantibiotics, in a total volume of 150 μL using standard 96 well plate.Plates were covered with sandwich covers (Model #CR1596 obtained fromEnzyScreen. Haarlam, The Netherlands) and 96 well plate cultures wereincubated at 37° C., 400 rpm for 24 hours. After 24 hours of production,all samples from each well were pelleted by centrifugation and thesupernatant collected for subsequent analytical measurement. Triplicatemicro-fermentations were performed for each strain.

For growth associated alanine micro-fermentations, glycerol stockpreparation and 16 hour overnight culture in SM10++ proceeded asdescribed above. After 16 hours of growth in SM10++ medium, 5 μL ofovernight culture was inoculated into 150 μL FGM3 with 40 mM phosphatecontaining appropriate antibiotic. Plates were covered with sandwichcovers (Model #CR1596 obtained from EnzyScreen, Haarlam, TheNetherlands) and 96 well plate cultures were incubated at 37° C., 400rpm for 24 hours. After 24 hours of production, OD600 was recorded, allsamples from each well were then pelleted by centrifugation and thesupernatant collected for subsequent analytical measurement. Triplicatemicro-fermentations were performed for each strain.

Micro-fermentation robustness evaluations were conducted as described inSupplemental Materials, Section 8.

1 L Fermentation Seeds

Single colony from transformation plate was inoculated into 5 mL LB withappropriate antibiotics and cultured at 37° C., 220 rpm for 16 hours,500 μL of the LB culture was inoculated into 50 mL SM10 media withappropriate antibiotics in square shake flask (CAT#: 25-214, GeneseeScientific, Inc. San Diego, Calif.), the culture was incubated at 37° C.with a shaking speed of 220 rpm for 24 hours, at which time OD600 isusually between 3 and 10, the culture was harvested by centrifugation at4000 rpm for 15 min, supernatant was discarded and cell culture wasnormalized to OD600=10 using SM10 media. For 1 L fermentation seed, 6 mLof normalized OD600=10 culture was added to 1.5 mL of 50% glycerol incryovials, and stored at −80° C.

1 L Fermentations

An Infors-HT Multifors (Laurel, Md., USA) parallel bioreactor system wasused to perform 1 L fermentations, including three gas connection massflow controllers configured for air, oxygen and nitrogen gases. Vesselsused had a total volume of 1400 mL and a working volume of up to 1 L.Online pH and pO2 monitoring and control were accomplished with Hamiltonprobes. Offgas analysis was accomplished with a multiplexed Blue-in-OneBlueSens gas analyzer (BlueSens. Northbrook. Ill., USA). Culturedensities were continually monitored using Optek 225 mm OD probes,(Optek, Germantown, Wis., USA). The system used was running IrisV6.0command and control software and integrated with a Seg-flowy automatedsampling system (Flownamics, Rodeo, Calif., USA), including FISP cellfree sampling probes, a Segmod 4800 and FlowFraction 96 well platefraction collector.

For the standardized 2-stage process with ˜10 gcdw/L biomass, tanks werefilled with 800 mL of FGM10 medium, with enough phosphate to target afinal E. coli biomass concentration ˜10 gcdw/L. Antibiotics were addedas appropriate. Frozen seed vials were thawed on ice and 7.5 mL of seedculture was used to inoculate the tanks. After inoculation, tanks werecontrolled at 37° C. and pH 6.8 using 5 M ammonium hydroxide and 1 Mhydrochloric acid as titrants. 10 M ammonium hydroxide was used for FIG.3G fermentation run. The following oxygen control scheme was used tomaintain the desired dissolved oxygen set point. First gas flow rate wasincreased from a minimum of 0.3 L/min of air to 0.8 L/min of air,subsequently, if more aeration was needed, agitation was increased froma minimum of 300 rpm to a maximum of 1000 rpm. Finally, if more oxygenwas required to achieve the set point, oxygen supplementation wasincluded using the integrated mass flow controllers. Starting glucoseconcentration was 25 g/L. A constant concentrated sterile filteredglucose feed (500 g/L) was added to the tanks at specified rate, i.e. 2g/h, once agitation reached 800 rpm. In cases where feed rate ordissolved oxygen content needed to be varied for robustness study,changes were made after cells entered stationary phase. Fermentationruns were extended for up to ˜50 hours after entry into stationary phaseand samples automatically withdrawn every 3 hours. Samples were savedfor subsequent analytical measurement.

In the case of growth associated fermentation processes, tanks werefilled with 800 mL of FGM10 medium with 40 mM phosphate, which was ingreat excess and ensured phosphate depletion doesn't happen for growthassociated fermentation processes. Antibiotics were added asappropriate. Frozen seed vials were thawed on ice and 7.5 mL of seedculture was used to inoculate the tanks. After inoculation, tanks werecontrolled at 37° C. and pH 6.8 using 5 M ammonium hydroxide and 1 Mhydrochloric acid as titrants. The following oxygen control scheme wasused to maintain the desired dissolved oxygen set point. First gas flowrate was increased from a minimum of 0.3 L/min of air to 0.8 L/min ofair, subsequently, if more aeration was needed, agitation was increasedfrom a minimum of 300 rpm to a maximum of 1000 rpm. Finally, if moreoxygen was required to achieve the set point, oxygen supplementation wasincluded using the integrated mass flow controllers. Starting glucoseconcentration was 25 g/L. A constant concentrated sterile filteredglucose feed (500 g/L) was added to the tanks at specified rate, i.e. 2g/h, once agitation reached 800 rpm. Feed rate and dissolved oxygenconcentration was set to desired values in the beginning, and maintainedthroughout the fermentation process. Fermentation runs were continuedfor up to˜50 hours and samples automatically withdrawn every 3 hours.Samples were saved for subsequent analytical analysis.

Analytical Methods

Sample standard curves for all compounds quantified are shown inSupplemental Materials. Section 10.

Glucose and Ethanol Quantification: A UPLC-R1 method was developed forthe simultaneous quantification of glucose and ethanol concentrations,using an Acquity H-Class UPLC integrated with a Waters 2414 RefractiveIndex (RI) detector (Waters Corp., Milford, Mass. USA). Chromatographicseparation was performed using a Bio-Rad Fast Acid Analysis HPLC Column(100×7.8 mm, 9 μm particle size: CAT#: #1250100. Bio-Rad Laboratories,Inc., Hercules, Calif.) at 65° C. 5 mM sulfuric acid was used as theeluent. The isocratic elution was as follows: 0-0.1 min, flow rateincreased from 0.4 mL/min to 0.42 mL/min, 0.1-12 min flow rate at 0.48mL/min. Sample injection volume was 10 μL. UPLC method development wascarried out using standard aqueous stock solutions of analytes. Peakintegration and further analysis was performed using MassLynx v4.1software. The linear range used for glucose was 1-10 g/L, for ethanolwas 1-20 g/L. Samples were diluted as needed to be within the accuratelinear range. Dilution was performed using ultrapure water.

Alanine Quantification: A reverse phase UPLC-MS/MS method was developedfor alanine. Chromatographic separation was performed using a RestekUltra AQ C18 column (150 mm×2.1 i.d., 3 μm; CAT#: 9178362, RestekCorporation, Bellefonte, Pa.) at 70° C. The following eluents were used:solvent A: H₂O, 0.2% formic acid and 0.05% ammonium (v/v); solvent B:MeOH, 0.2% formic acid and 0.05% ammonium (v/v). The gradient elutionwas as follows: 0-0.1 min isocratic 5% B, flow rate increased from 0.65mL/min to 0.75 mL/min; 0.1-0.3 min, linear from 5% to 95% B at 0.75mL/min; 0.3-0.9 min isocratic 95% B at 0.75 mL/min; and 0.9-1.2 minlinear from 95% to 5% B at 0.75 mL/min; 1.2-1.3 min isocratic 5% B at0.75 mL/min. Sample injection volume was 5 μL. UPLC method developmentwas carried out using standard aqueous stock solutions of analyte.Separations were performed using an Acquity H-Class UPLC integrated witha Xevo™ TQD Mass spectrometer (Waters Corp., Milford. Mass. USA). MS/MSparameters including MRM transitions were tuned for each analyte and arelisted in Table 22. Alanine (2.3-13C2, 99%) was used as internalstandard for alanine at a concentration of 5 mg/L. Peak integration andfurther analysis was performed using MassLynx v4.1 software. The linearrange for alanine was 1-100 mg/L. Samples were diluted as needed to bewithin the accurate linear range. Dilution was performed using ultrapurewater, and the final 10-fold dilution was performed using solvent A,with 5 mg/l of C13 alanine (2,3-13C2, 99%).

Mevalonic Acid Quantification: A reverse phase UPLC-TUV method wasdeveloped for the simultaneous quantification of mevalonic acid andmevalonolactone. Chromatographic separation was performed using a RestekUltra AQ C18 column (150 mm×2.1 i.d., 3 μm; CAT#: 9178362, RestekCorporation, Bellefonte, Pa.) at 30° C. 20 mM phosphoric acid was usedas the eluent. The isocratic elution was as follows: 0-3 min isocraticat 1 mL/min. Sample injection volume was 10 μL. Absorbance was monitoredat 210 nm. UPLC method development was carried out using standardaqueous stock solutions of analytes. Separations were performed using anAcquity H-Class UPLC (Waters Corp., Milford, Mass. USA). Peakintegration and further analysis was performed using MassLynx v4.1software. The linear range for mevalonic acid and mevalonolactone were0.01-0.1 g/L. Samples were diluted as needed to be within the accuratelinear range. Mevalonic acid diluted in 20 mM phosphoric acid wouldspontaneously convert to mevalonolactone⁸⁰, thus, quantification of bothmevalonic acid and mevalonolactone was necessary for fermentationsamples. Mevalonic acid and mevalonolactone standards were preparedfresh each time, and ran immediately on UPLC. Dilution was performedusing ultrapure water, and the final 10-fold dilution was performedusing 20 mM phosphoric acid.

Alanine Stereoisomer Quantification: A reverse phase UPLC-TUV method wasdeveloped for the simultaneous quantification and differentiation ofL-/D-alanine. Chromatographic separation was performed using a Chirex3126 (D)-penicillamine column (150×4.6 mm, 5 μm; Phenomenex Inc.,Torrance, Calif.) at 50° C. 2 mM Copper Sulfate was used as the eluent.The isocratic elution was as follows: 0-10 min at 0.75 mL/min. Sampleinjection volume was 10 μL. Absorbance was monitored at 254 nm. UPLCmethod development was carried out using standard aqueous stocksolutions of analytes. Separations were performed using an AcquityH-Class UPLC (Waters Corp., Milford, Mass. USA). Peak integration andfurther analysis was performed using MassLynx v4.1 software. The linearrange for L-/D-alanine was 0.1-1 g/L. Samples were diluted as needed tobe within the accurate linear range. Dilution was performed usingultrapure water.

Supplemental Materials

TABLE 1 Combinatorial complexity of metabolic networks. Number ofExperiments Entire E. coli Combination # Gene Network Reduced CentralMetabolism Network 1 4500 ~45 (Glycolysis, TCA, PPP and ETC genes only)2 1.0 × 10⁶    990 3  1.5 × 10¹⁰  14,190 4  1.7 × 10¹³ 148,995 5  1.5 ×10¹⁶ 1.2 × 10⁶

Section 1: Phosphate Promoters

Phosphate promoter sequences were obtained from the EcoCyc database⁸¹for PhoB regulated promoters (https://ecocyc.org/, Table 2). We soughtto evaluate not only the relative strength of promoters previouslycharacterized to respond to phosphate depletion, but in addition therelative leakiness in phosphate rich conditions. To this aim weconstructed a set of fluorescent reporter plasmids. We cloned theultraviolet excitable GFPuv gene behind a set of 12 phosphate dependentpromoters, in the pSMART-HC-Kan (Lucigen. WI) backbone. These reporterstrains were evaluated in a 2-stage micro-fermentation protocol in anm2p-labs Biolector™. Results are illustrated in FIG. 7. The ugpB genepromoter was often chosen for high level tightly controlled expressionwhen expression cassettes were chromosomally integrated or for theinducible expression of guide arrays.

Insulators⁸² were added to both 5′ and 3′ end of a subset of phosphatepromoters (Table 3) to help with consistent performance in differentsequence contexts. To reduce read-through transcription, a uniqueterminator was added to the 3′ end of each insulated promoter.Terminator sequences were fromhttp://parts.igem.org/Terminators/Catalog. Insulated phosphate promoterswere similarly characterized using GFPuv expression in a m2p-labsBiolector™ (FIG. 8).

TABLE 2Phosphate inducible promoter sequences evaluated; the ribosomal binding siteis underlined, and the start codon of the gene (GFPuv) is shown in green.Promoter SEQ Name Sequence ID NO ugpBpTCTTTCTGACACCTTACTATCTTACAAATGTAACAAAAAAGTTATTTTTCTGTAATTCGA  1GCATGTCATGTTACCCCGCGAGCATAAAACGCGTGTGTAGGAGGATAATCTATG yibDpGTGCGTAATTGTGCTGATCTCTTATATAGCTGCTCTCATTATCTCTCTACCCTGAAGTGAC  2TCTCTCACCTGTAAAAATAATATCTCACAGGCTTAATAGTTTCTTAATACAAAGCCTGTAAAACGTCAGGATAACTTCTGTGTAGGAGGATAATCTATG phoApCGATTACGTAAAGAAGTTATTGAAGCATCCTCGTCAGTAAAAAGTTAATCTTTTCAACA  3GCTGTCATAAAGTTGTCACGGCCGAGACTTATAGTCGCTTTGTTTTTATTTTTTAATGTATTTGTAGTGTAGGAGGATAATCTATGGCTAGCAAAGGAGAAGAACTTTTCACATG phoBpGCCACGGAAATCAATAACCTGAAGATATGTGCGACGAGCTTTTCATAAATCTGTCATAA  4ATCTGACGCATAATGACGTCGCATTAATGATCGCAACCTATTTATTGTGTAGGAGGATAATCTATGGCTAGCAAAGGAGAAGAACTTTTCACATG amnpAGACAGTCAACGCGCTTGATAGCCTGGCGAAGATCATCCGATCTTCGCCTTACACTTTTG  5TTTCACATTTCTGTGACATACTATCGGATGTGCGGTAATTGTATAGGAGGATAATCTATG ydfHpGCTATGCCGGACTGAATGTCCACCGTCAGTAATTTTTATACCCGGCGTAACTGCCGGGTT  6ATTGCTTGTCACAAAAAAGTGGTAGACTCATGCAGTTAACTCACTGTGTAGGAGGATAA TCTATG mipApCATCCATAAATTTTGCATAATTAATGTAAAGACCAGGCTCGCCAGTAACGCTAAATTCA  7TTTGGCTGTAAGCGCGGTGTCATCCGCGTCAGGAAAATTAAACAGTTACTTTAAAAAATGAAAACGTAAAAAGGTTGGGTTTCGATGTATTGACGGGTAAACTTTGTCGCCCGCTAAACATTTGTTTGTGTAGGAGGATAATCTATG phoHpAATCCTGCTGAAAGCACACAGCTTTTTTCATCACTGTCATCACTCTGTCATCTTTCCAGT  8AGAAACTAATGTCACTGAAATGGTGTTTTATAGTTAAATATAAGTAAATATATTGTTGCAATAAATGCGAGATCTGTTGTACTTATTAAGTAGCAGCGGAAGTTCGTGTAGGAGGATAA TCTAT yhjCpCTACAGAGATGACGTGTAGAAAATAGTTACCGATATAAATAGTTACAGCTAAACGCCTG  9AAATTACATGTCGAGGGCACTATTTAAAACAATTTTGAGGATTTCCTTATATTGGTGGTTAGTACGCATGCAATTAAAAATGAAATTCCGCGACCACAAGCCAAAATAACAAACGGCAAGGAGACAAAAATAAGCACAAATAGCCAACACGTCCTCTGTTCACTTTAAAGGGAATCGCTGAAAAATACGCTCTGTTTAAGGGGATTCACCTTTCTCAGAAAGCTATTCCGCCCTTTTCCTGCTGAGAAATCGCCACATTCGGCATGACAACATTGTGAAAGTGTAGGAGGATAATC TATG phoUpACCGAACTGAAGCAGGATTACACCGTGGTGATCGTCACCCACAACATGCAGCAGGCTGC 10GCGTTGTTCCGACCACACGGCGTTTATGTACCTGGGCGAATTGATTGAGTTCAGCAACACGGACGATCTGTTCACCAGTGTAGGAGGATAATCTATG pstSpAAGACTTTATCTCTCTGTCATAAAACTGTCATATTCCTTACATATAACTGTCACCTGTTTG 11TCCTATTTTGCTTCTCGTAGCCAACAAACAATGCTTTATGAGTGTAGGAGGATAATCTATGGCTAGCAAAGGAGAAGAACTTTTCACATG phoEpAGCATGGCGTTTTGTTGCGCGGGATCAGCAAGCCTAGCGGCAGTTGTTTACGCTTTTATT 12ACAGATTTAATAAATTACCACATTTTAAGAATATTATTAATCTGTAATATATCTTTAACAATCTCAGGTTAAAAACTTTCCTGTTTTCAACGGGACTCTCCCGCTGGTGTAGGAGGATAA TCTATG

TABLE 3Insulated promoter sequences. Insulator sequences are italicized. -35 and -10boxes are highlighted in bold and underlined. SEQ Insulated PromoterSequence ID NO BBa_B0015_IN_yibDpCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTT 13TTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATACACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACGTGCGTAATTGTGCTGATCTCTTATATAGCTGCTCTCATTATCTCTCTACCCTGAA GTGACT CTCTCACCTGTAAAAATAATATCTCACAGGCT TAATA GTTTCTTAATACAAAGCCTGTAAAACGTCAGGATAACTTCTATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTT TGTTTAACTTTBBa_B1002_IN_phoBp CGCAAAAAACCCCGCTTCGGCGGGGTTTTTTCGCACCGTCTCCATCGCTTGCC14 CAAGTTGTGAAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACGCCACGGAAATCAATAACCTGAAGATATGTGCCG ACGAGCTT TTCATAAATCTGTCATAAATCTGACG CATAAT GACGTCGCATTAATGATCGCAACCTATTTATTATATTCAGGGAGACCACAACGGTTTCCCTCTACCAATAATTTTGTTTAACTTT BBa_B1004_IN_mipApCGCCGAAAACCCCGCTTCGGCGGGGTTTTGCCGCACGTCTCCATCGCTTGCC 15CAAGTTGTGAAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACCATCCATAAATTTTGCATAATTAATGTAAAGACCAGGCTCGCCAGTAACGCTAAATTCATTTGGCTGTAAGCGCGGTGTCATCCGCGTCAGGAAAATTAAACAGTTACTTTAAAAAATGAAAACGTAAA AAGGTT GGGTTTCGATGTATTGACGG GTAAAC TTTGTCGCCCGCTAAACATTTGTTTATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTTT BBa_B1006_IN_phoUpAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTACGTCTCCATCGC 16TTGCCCAAGTTGTGAAGCACAGCTAACACCACGTCGTCCCTATGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACACCGAACTGAAGCAGGATTACACCGTGGTGATCGTCACCCACAACATGCAGCAGGCTGCGCGTTGTTCCGACCACA CGG CGTTTATGTACCTGGGCGAATT GATTGA GTTCAGCAACACGGACGATCTGTTCACCAATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTT TBBa_B1010_IN_phoHp CGCCGCAAACCCCGCCCCTGACAGGGCGGGGTTTCGCCGCACGTCTCCATCG17 CTTGCCCAAGTTGTGAAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACAATCCTGCTGAAAGCACACAGCTTTTTT CATCACTGTCATCACTCTGTCA TCTTTCCAGTAGAAAC TAATGT CACTGAAATGGTGTTTTATAGTTAAATATAAGTAAATATATTGTTGCAATAAATGCGAGATCTGTTGTACTTATTAAGTAGCAGCGGAAGTTCATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTTT

Section 2: Constitutive Promoters

A set of constitutive insulated promoters of varying strength were usedfor constitutive expression and taken directly from Davis et al.,including the proA, proB, proC, proD promoters⁸² and HCEp promoter⁸³.Insulator was added to 5′ and 3′ of HCEp promoter. Similar to insulatedphosphate promoters, a unique terminator was added to the 5′ end ofconstitutive promoters. These were used to drive constitutive pathwayexpression in growth associated production strains as well as to makestrain modifications where constitutive heterologous gene expression wasappropriate. These promoter sequences are given in Table 4 below andpromoter characterized using GFPuv expression (FIG. 9).

TABLE 4 Constitutive promoter sequences. Promoter Sequence SEQ ID NOBBa_B1004_proA CGCCGAAAACCCCGCTTCGGCGGGGTTTTGCCGCACGTC 18TCCATCGCTTGCCCAAGTTGTGAAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTAGGCTATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAAT TTTGTTTAACTTT BBa_B1006_proBAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTT 19ACGTCTCCATCGCTTGCCCAAGTTGTGAAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGCGCATGCATAAGGCTCGTAATATATATTCAGGGAGACCACAACGGTTTCCCTCTACAA ATAATTTTGTTTAACTTTBBa_B1010_proC CGCCGCAAACCCCGCCCCTGACAGGGCGGGGTTTCGCC 20GCACGTCTCCATCGCTTGCCCAAGTTGTGAAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTATGATATATTCAGGGAGACCACAACGGTTTCCCTCTAC AAATAATTTTGTTTAACTTTBBa_B1002_proD CGCAAAAAACCCCGCTTCGGCGGGGTTTTTTCGCACGTC 21TCCATCGCTTGCCCAAGTTGTGAAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTATAATATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAAT TTTGTTTAACTTT BBa_B0015_IN_HCEpCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGAC 22TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATACACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACCTCCTTCACAGATTCCCAATCTCTTGTTAAATAACGAAAAAGCATCAATTAAAACCCATGTCTTTCTATATTCCAGCAATGTTTTATAGGGGACATATTGATGAAGATGGGTATCACCTTAGTGAATTGCTATAAGCTGCTCTTTTTTGTTCGTGATATACTGATAAATTGAATTTTCACACTTCATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTTT

Section 3: Chromosomally Modified Host Strains

FIG. 11 depicts each chromosomal modification Strains utilized and/orconstructed for this study are listed in Table 5. Tables 6 and 7 listsoligonucleotides and synthetic DNA sequences used for strainconstruction and/or confirmation. FIG. 12 and FIG. 13A-E show growthrates and glucose distribution during growth for control strains in 1 Lfermentation.

TABLE 5 List of chromosomally modified strains. Strain Genotype SourceBW25113 (wt) F-, λ-, Δ(araD-ataB)567, lacZ4787(del)(::rrnB-3), rph-1,CGSC Δ(rhaD-rhaB)568, hsdR514 JW3197-1 BW25113, sspB756(del)::.kan ⁵³Bwapldf BW25113, ΔackA-pta, ΔpoxB, ΔpflB, ΔldhA, ΔadhE ³⁹ DLF_0001 BWapldf, ΔiclR, ΔarcA this study DLF_0002  BWapldf, ΔiclR, ΔarcA,ΔsspB::frt this study DLF_0025  DLF_0002,Δcas3::tm-ugpb-sspB-pro-casA(N2S) this study DLF_0028  DLF_0025,fabl-DAS + 4-gentR this study DLF_0031  DLF_0025, lpd-DAS + 4-gentR thisstudy DLF_0038  DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + 4-bsdR thisstudy DLF_0039  DLF_0025, fabI-DAS + 4-gentR, gltA-DAS + 4-zeoR thisstudy DLF_0040  DLF_0025, fabI-DAS + 4-gentR, zwf-DAS + 4-bsdR thisstudy DLF_0041  DLF_0025, lpd-DAS + 4-gentR, gltA-DAS + 4-zeoR thisstudy DLF_0042  DLF_0025, lpd-DAS + 4-gentR, udhA-DAS + 4-bsdR thisstudy DLF_0043  DLF_0025, gltA-DAS + 4-zeoR this study DLF_0044 DLF_0025, gltA-DAS + 4-zeoR, zwf-DAS + 4-bsdR this study DLF_0045 DLF_0025, gltA-DAS + 4-zeoR, udhA-DAS + 4-bsdR this study DLF_0046 DLF_0025, fabI-DAS + 4-gentR, gltA-DAS + this study 4-zeoR, zwf-DAS +4-bsdR DLF_0047  DLF_0025, fabI-DAS + 4-aentR, gltA-DAS + this study4::zeoR, udhA-DAS + 4-bsdR DLF_0048  DLF_0025, lpd-DASH + 4-gentR,gltA-DAS + this study 4-zeoR, zwf-DAS + 4-bsdR DLF_0049  DLF_0025,lpd-DAS + 4-gentR, gltA-DAS + this study 4-zeoR, udhA-DAS + 4-bsdRDLF_0165  DLF_0025, lpd-DAS + 4-gentR, zwf-DAS + 4-bsdR this studyDLF_0763  DLF_0025, udhA-DAS + 4-bsdR this study DLF_01002 DLF_0025,zwf-DAS + 4-bsdR this study DLF_01517 DLF_0012, Δcas3::pro-casA(N2S)this study DLF_01530 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + this study4-bsdR, zeoR-proDp-gapN-zeoR DLF_01531 DLF_0025, fabI-DAS + 4-gentR,udhA-DAS + this study 4-bsdR, gltA-DAS + 4-purR DLF_01532 DLF_0025,fabI-DAS + 4-gentR, udhA-DAS + this study 4-bsdR, gapA-DAS +4-zeoR-proDp-gapN DLF_01533 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS +4-bsdR, this study gapA-DAS + 4-zeoR-proDp-gapN, gltA-DAS + 4-purRDLF_01536 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + this study 4-bsdR,zeoR-proDp-gapN, gltA-DAS + 4-purR DLF_01537 DLF_0025, fabI-DAS +4-gentR, udhA-DAS + this study 4-bsdR, gapA-DAS + 4-zeoR DLF_01538DLF_0025, fabI-DAS + 4-gentR, gltA-DAS + this study 4-zeoR, udhA-DAS +4-bsdR, gapA-DAS + 4-zeoR

TABLE 6 Oligonucleotides utilized for strain construction Oligo SequenceSEQ ID NO ilcR_tetA_F TAACAATAAAAATGAAAATGATTTCCACGATACAGAAA 23AAAGAGACTGTCATCCTAATTTTTGTTGACACTCTATC ilcR_sacB_RTGCCACTCAGGTATGATGGGCAGAATATTGCCTCTGCCC 24GCCAGAAAAAGATCAAAGGGAAAACTGTCCATATGC iclR_500up CCCGACAGGGATTCCATCTG 25iclR_500dn TATGACGACCATTTTGTCTACAGTTC 26 arcA_tetA_FGGACTTTTGTACTTCCTGTTTCGATTTAGTTGGCAATTTA 27GGTAGCAAACTCCTAATTTTTGTTGACACTCTATC arcA_sacB_RATAAAAACGGCGCTAAAAAGCGCCGTTTTTTTTGACGGT 28GGTAAAGCCGAATCAAAGGGAAAACTGTCCATATGC arcA_500up CCTGACTGTACTAACGGTTGAG29 arcA_500dn TGACTTTTATGGCGTTCTTTGTTTTTG 30 sspB_kan_FCTGGTACACGCTGATGAACACC 31 sspB_kan_R CTGGTCATTGCCATTTGTGCC 32sspB_conf_F CAATCAGAGCGTTCCGACCC 33 sspB_conf_R GTACGCAGTTTGCCAACGTG 34cas3_tetA_F AATAGCCCGCTGATATCATCGATAATACTAAAAAAAACAG 35GGAGGCTATTATCCTAATTTTTGTTGACACTCTATC cas3_sacB_RTACAGGGATCCAGTTATCAATAAGCAAATTCATTTGTTCT 36CCTTCATATGATCAAAGGGAAAACTGTCCATATGC cas3_conf_FCAAGACATGTGTATATCACTGTAATTC 37 cas3_500dn GCGATTGCAGATTTATGATTTGG 38fabl_conf_F GCAAAATGCTGGCTCATTG 39 gapA_conf_F GAACTGAATGGCAAACTGACTG 40gapA_500dn TGGGGATGATCGACCACA 41 gltA_conf_F TATCATCCTGAAAGCGATGG 42lpd_conf_F ATCTCACCGTGTGATCGG 43 udhA_conf_F CAAAAGAGATTCTGGGTATTCACT 44zwf_conf_F CTGCTGGAAACCATGCG 45 zwf_500dn AGAGCATGTCGTTATAGGAGGTGAT 46ampR_intR AGTACTCAACCAAGTCATTCTG 47 bsdR_intR GAGCATGGTGATCTTCTCAGT 48gentR_intR GCGATGAATGTCTTACTACGGA 49 purR_intR GTCGCTGGGTAATCTGCAA 50tetA_intR ATCAACGCATATAGCGCTAGCAG 51 zeoR_intR ACTGAAGCCCAGACGATC 52

TABLE 7 Synthetic DNA utilized for strain construction. SEQ ID NOtetA-sacB Cassette TCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCTA53 TCAGTGATAGAGAAAAGTGAAATGAATAGTTCGACAAAGATCGCATTGGTAATTACGTTACTCGATGCCATGGGGATTGGCCTTATCATGCCAGTCTTGCCAACGTTATTACGTGAATTTATTGCTTCGGAAGATATCGCTAACCACTTTGGCGTATTGCTTGCACTTTATGCGTTAATGCAGGTTATCTTTGCTCCTTGGCTTGGAAAAATGTCTGACCGATTTGGTCGGCGCCCAGTGCTGTTGTTGTCATTAATAGGCGCATCGCTGGATTACTTATTGCTGGCTTTTTCAAGTGCGCTTTGGATGCTGTATTTAGGCCGTTTGCTTTCAGGGATCACAGGAGCTACTGGGGCTGTCGCGGCATCGGTCATTGCCGATACCACCTCAGCTTCTCAACGCGTGAAGTGGTTCGGTTGGTTAGGGGCAAGTTTTGGGCTTGGTTTAATAGCGGGGCCTATTATTGGTGGTTTTGCAGGAGAGATTTCACCGCATAGTCCCTTTTTTATCGCTGCGTTGCTAAATATTGTCACTTTCCTTGTGGTTATGTTTTGGTTCCGTGAAACCAAAAATACACGTGATAATACAGATACCGAAGTAGGGGTTGAGACGCAATCGAATTCGGTATACATCACTTTATTTAAAACGATGCCCATTTTGTTGATTATTTATTTTTCAGCGCAATTGATAGGCCAAATTCCCGCAACGGTGTGGGTGCTATTTACCGAAAATCGTTTTGGATGGAATAGCATGATGGTTGGCTTTTCATTAGCGGGTCTTGGTCTTTTACACTCAGTATTCCAAGCCTTTGTGGCAGGAAGAATAGCCACTAAATGGGGCGAAAAAACGGCAGTACTGCTCGGATTTATTGCAGATAGTAGTGCATTTGCCTTTTTAGCGTTTATATCTGAAGGTTGGTTAGTTTTCCCTGTTTTAATTTTATTGGCTGGTGGTGGGATCGCTTTACCTGCATTACAGGGAGTGATGTCTATCCAAACAAAGAGTCATCAGCAAGGTGCTTTACAGGGATTATTGGTGAGCCTTACCAATGCAACCGGTGTTATTGGCCCATTACTGTTTGCTGTTATTTATAATCATTCACTACCAATTTGGGATGGCTGGATTTGGATTATTGGTTTAGCGTTTTACTGTATTATTATCCTGCTATCGATGACCTTCATGTTAACCCCTCAAGCTCAGGGGAGTAAACAGGAGACAAGTGCTTAGTTATTTCGTCACCAAATGATGTTATTCCGCGAAATATAATGACCCTCTTGATAACCCAAGAGCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAACCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAGGCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACCTTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCACTACAAACAATCAACGTTTGCGCCAAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATATTGACTACCGCAAGCAGTGTGACCGTGTGCTTCTCAAATGCCTGATTCAGGCTGTCTATGTGTGACTGTTGAGCTGTAACAAGTTGTCTCAGGTGTTCAATTTCATGTTCTAGTTGCTTTGTTTTACTGGTTTCACCTGTTCTATTAGGTGTTACATGCTGTTCATCTGTTACATTGTCGATCTGTTCATGGTGAACAGCTTTAAATGCACCAAAAACTCGTAAAAGCTCTGATGTATCTATCTTTTTTACACCGTTTTCATCTGTGCATATGGACAGTTTTCCCTT TGAT ΔiclR-cureAAATGATTTCCACGATACAGAAAAAAGAGACTGTCATGGGCAGAATATTGC 54CTCTGCCCGCCAGAAAAAG ΔarcA-cureCTGTTTCGATTTAGTTGGCAATTTAGGTAGCAAACTCGGCTTTACCACCGTC 55AAAAAAAACGGCGCTTTT Δcas3-pro-casACAAGACATGTGTATATCACTGTAATTCGATATTTATGAGCAGCATCGAAAAA 56TAGCCCGCTGATATCATCGATAATACTAAAAAAACAGGGAGGCTATTACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATCTTTCTGACACCTTACTATCTTACAAATGTAACAAAAAAGTTATTTTTCTGTAATTCGAGCATGTCATGTTACCCCGCGAGCATAAAACGCGTGTGTAGGAGGATAATCTTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCCATATGAAGGAGAACAAATGAATTTGCTTATTGATAACTGGATCCCTGTACGCCCGCGAAACGGGGGGAAAGTCCAAATCATAAATCTGCA ATCGCTATACΔcas3::ugBp-sspB-pro-casACAAGACATGTGTATATCACTGTAATTCGATATTTATGAGCAGCATCGAAAAA 57TAGCCCGCTGATATCATCGATAATACTAAAAAAACAGGGAGGCTATTACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATCTTTCTGACACCTTACTATCTTACAAATGTAACAAAAAAGTTATTTTTCTGTAATTCGAGCATGTCATGTTACCCCGCGAGCATAAAACGCGTGTGTAGGAGGATAATCTATGGATTTGTCACAGCTAACACCACGTCGTCCCTATCTGCTGCGTGCATTCTATGAGTGGTTGCTGGATAACCAGCTCACGCCGCACCTGGTGGTGGATGTGACGCTCCCTGGCGTGCAGGTTCCTATGGAATATGCGCGTGACGGGCAAATCGTACTCAACATTGCGCCGCGTGCTGTCGGCAATCTGGAACTGGCGAATGATGAGGTGCGCTTTAACGCGCGCTTTGGTGGCATTCCGCGTCAGGTTTCTGTGCCGCTGGCTGCCGTGCTGGCTATCTACGCCCGTGAAAATGGCGCAGGCACGATGTTTGAGCCTGAAGCTGCCTACGATGAAGATACCAGCATCATGAATGATGAAGAGGCATCGGCAGACAACGAAACCGTTATGTCGGTTATTGATGGCGACAAGCCAGATCACGATGATGACACTCATCCFGACGATGAACCTCCGCAGCCACCACGCGGTGGTCGACCGGCATTACGCGTTGTGAAGTAATTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCCATATGAAGGAGAACAAATGAATTTGCTTATTGATAACTGGATCCCTGTACGCCCGCGAAACGGGGGGAAAGTCCAAATCATAAATCTGCAATCGCTATAC fabI-DAS+4-gentRCTATTGAAGATGTGOGTAACTCTGCGCCATTCCTGTGCTCCGATCTCTCTGC 58CGOTATCTCCGGTGAAGTGGTCCACGTTGACGGCGGTTTCAGCATTGCTGCAATGAACGAACTCGAACTGAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCGAATCCATGTGGGAGTTTATTCTTGACACAGATATTTATGATATAATAACTGAGTAAGCTTAACATAAGGAGGAAAAACATATGTTACGCAGCAGCAACGATGTTACGGAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCACTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAAGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCGTTCTGTTGGTAAAGATGGGCGGCGTTCTGCCGCCCGTTATCTCTGTTATACCTTTCTGATATTTGTTATCGCCGATCCGTCTTTCTCCCCTTCCCGCCTTGCGTCAGG gapA-DAS+4-zeoR-proDp-gapNTCTCCAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGT 59CTTGATTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAACTTTAAAATTAAAGAGGTATATATTAATGACTAACCAATATAAGAATTACGTAAATGGGGAGTGGAAGCTTTCGGAGAATGAAATTAAGATCTATGAACCAGCCAGTGGGGCGGAATTGGGGTCAGTCCCGGCAATGTCCACTGAAGAAGTFGACTATGTCTACGCCTCGGCCAAAAAAGCGCAGCCAGCATGGCGCTCGCTTTCCTATATTGAGCGTGCGGCTTATTTGCACAAAGTCGCAGACATCCTGATGCGTGACAAGGAGAAAATTGGAGCGGTATTGTCCAAGGAAGTAGCGAAAGGCTACAAATCCGCAGTATCGGAGGTCGTCCGCACCGCCGAGATTATTAATTATGCGGCCGAAGAAGGGCTTCGCATGGAGGGTGAGCTTCTTGGAGGGCGGCAGTTTTGAGGCGGCATCCAAGAAAAAAATCGCTGTCGTCCGTCGCGAGCCGGTGGGACTTCTGCTTGCTATTAGTCCGTTCAATTACCCCGTGAATCTGGCCGGCTCCAAGATTCCCCTGCACTGATCGCGGGCAATGTAATCGCTTTTAAACCACCGACCCAAGGATCGATTAGTGGACTTCTTTTAGCGGAGGCGTTTGCGGAGGCAGGTCTTCCAGCCGGCGTATTCAATACCATCACGGGGCGTGGAAGTGAAATCGGGGATTACATCGTGGAGCACCAGGCAGTAAATTTCATCAACTTCACGGGTTCCACGGGGATCGGGGAGCGTATCGGTAAGATGGCTGGGATGCGTCCGATCATGTTGGAACTTGGCGGCAAGGATAGTGCGATTGTGCTGGAAGACGCAGACTTGGAATTCACAGCTAAAAACATTATCGCTGGAGCCTTCGGGTATAGTGGTCAACGTTGGACGGCAGTTAAGCGCGTTCTTGTTATGGAAAGTGTCGCGGATGAATTGGTCGAGAAGATTCGCGAGAAAGTGTTAGCTCTTACGATTGGAAATCCAGAGGACGATGCTGACATCACTCCATTGATCGACACGAAATCCGCGGATTACGTCGAGGGGCTGATCAACGACGCGAACGATAAGGGAGCAGCGGCTTTGACCGAGATCAAACGCGAGGGGAACCTGATCTGCCCGATTCTTTTTGACAAAGTCACAACTGACATGCGCTTGGCATGGGAAGAACCCTTCGGCCCAGTCTTGCCTATTATCCGCGTTACTAGCGTAGAGGAAGCAATTGAAATTTCCAATAAATCCGAATATGGGTTGCAAGCGAGTATCTTTACTAACGATTTTCCACGTGCCTTTGGTATTGCGGAACAGTTAGAAGTCGGGACAGTTCACATCAACAACAAGACGCAGCGCGGGACAGATAACTTCCCCTTTTTGGGAGCAAAGAAGTCTGGGGCTGGAATCCAAGGGGTGAAATACTCCATCGAAGCCATGACGACGGTGAAGAGCGTTGTTTTTGACATCAAGTAAAACATAAGGAGGAAAAACAGATGGCGAAACTGACCTCGGCGGTTCCGGTTCTGACGGCACGTGATGTGGCGGGCGGGGTTGAATTTTGGACGGATCGTCTGGGCTTCAGTCGTGATTTTGTGGAAGATGACTTCGCAGGCGTGGTTCGCGATGACGTCACCCTGTTTATTTCCGCAGTTCAGGATCAAGTCGTGGCGGACAACACGCTGGCTTGGGTGTGGGTTCGTGGCCTGGATGAACTGTATGCGGAATGGAGCGAAGTTGTCTCTACCAATTTCCGTGACGCGAGCGGTCCGGCCATGACGGAAATCGGCGAACAGCCGTGGGCTCGCGAATTTGCTCTGCGTGACCCGGCTGGCAACTGTGTCCATTTCGTGGCTGAAGAACAAGATTGAGTTGAGATGACACTGTGATCTAAAAAGAGCGACTTCGGTCGCTCTTTTTTTTACCTGA gapA-zeoR-proDp-gapNACGAAACCGGTTACTCCAACAAAGTTCTGGACCTGATCGCTCACATCTCCAA 60ATGATTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAACTTTAAAATTAAAGAGGTATATATTAATGACTAAGCAATATAAGAATTACGTAAATGGGGAGTGGAAGCTTTCGGAGAATGAAATTAAGATCTATGAACCAGCCAGTGGGGCGGAATTGGGGTCAGTCCCGGCAATGTCCACTGAAGAAGTTGACTATGTCTACGCCTCGGCCAAAAAAGCGCAGCCAGCATGGCGCTCGCTTTCCTATATTGAGCGTGCGGCTTATTTGCACAAAGTCGCAGACATCCTGATGCGTGACAAGGAGAAAATTGGAGCGGTATTGTCCAAGGAAGTAGCGAAAGGCTACAAATCCGCAGTATCGGAGGTCGTCCGCACCGCCGAGATTATTAATTATGCGGCCGAAGAAGGGCTTCGCATGGAGGGTGAGGTCTTGGAGGGCGGCAGTTTTGAGGCGGCATCCAAGAAAAAAATCGCTGTCGTCCGTCGCGAGCCGGTGGGACTTGTGCTTGCTATTAGTCCGTTCAATTACCCCGTGAATCTGGCCGGCTCCAAGATTGCCCCTGCACTGATCGCGGGCAATGTAATCGCTTTTAAACCACCGACCCAAGGATCGATTAGTCGACTTCTTTTAGCGGAGGCGTTTGCGGAGGCAGGTCTTCCAGCCGGCGTATTCAATACCATCACGGGGCGTGGAAGTGAAATCGGGGATTACATCGTGGAGCACCAGGCAGTAAATTTCATCAACTTCACGGGTTCCACGGGGATCGGGGAGCGTATCGGTAAGATGGCTGGGATGCGTCCGATCATGTTGGAACTTGGCGGCAAGGATAGTGCGATTGTGCTGGAAGACGCAGACTTGGAATTGACAGCTAAAAACATTATCGCTGGAGCCTTCGGGTATAGTGGTCAACGTTGCACGGCAGTTAAGCGCGTTCTTGTTATGGAAAGTGTCGCGGATGAATTGGTCGAGAAGATTCGCGAGAAAGTGTTAGCTCTTACGATTGGAAATCCAGAGGACGATGCTGACATCACTCCATTGATCGACACGAAATCCGCGGATTACGTCGAGGGGCTGATCAACGACGCGAACGATAAGGGAGCAGCGGCTTTGACCGAGATCAAACGCGAGGGGAACCTGATCTGCCCGATTCTTTTTGACAAAGTCACAACTGACATGCGCTTGGCATGGGAAGAACCCTTCGGCCCAGTCTTGCCTATTATCCGCGTTACTAGCGTAGAGGAAGCAATTGAAATTTCCAATAAATCCGAATATGGGTTGCAAGCGAGTATCTTTACTAACGATTTTCCACGTGCCTTTGGTATTGCGGAACAGTTAGAAGTCGGGACAGTTCACATCAACAACAAGACGCAGCGCGGGACAGATAACTTCCCCTTTTTGGGAGCAAAGAAGTCTGGGGCTGGAATCCAAGGGGTGAAATACTCCATCGAAGCCATGACGACGGTCAAGAGCGTTGTTTTTGACATCAAGTAAAACATAAGGAGGAAAAACAGATGGCGAAACTGACCTCGGCGGTTCCGGTTCTGACGGCACGTGATGTGGCGGCCGCCGTTGAATTTTGGACGGATCGTCTGGGCTTCAGTCGTGATTTTGTGGAAGATGACTTCGCAGGCGTGGTTCGCGATGACGTCACCCTGTTTATTTCCGCAGTTCAGGATCAAGTCGTGCCGGACAACACGCTGGCTTGGGTGTGGGTTCGTGGCCTGGATGAACTGTATGCGGAATGGAGCGAAGTTGTCTCTACCAATTTCCGTGACGCGAGCGGTCCGGCCATGACGGAAATCGGCGAACAGCCGTGGGGTCGCGAATTTGCTCTGCGTGACCCGGCTGGCAACTGTGTCCATTTCGTGGCTGAACAACAAGATTGAGTTGAGATGACACTGTGATCTAAAAAGAGCGACTTCGGTCGCTCTTTTTTTTACCTGA gapA-DAS+4-zeoRTCTACCGATTTCAACGGCGAAGTTTGCACTTCCGTGTTCGATGCTAAAGCTG 61GTATCGCTCTGAACGACAACTTCGTGAAACTGGTATCCTGGTACGACAACGAAACCGGTTACTCCAACAAAGTTCTGGACCTGATCGCTCACATCTCCAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTGATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAATGGCGAAACTGACCTCGCCGGTTCCGGTTCTGACGGCACGTGATGTGGCGGGCGCGGTTGAATTTTGGACGGATCGTCTGGGCTTGAGTCGTGATTTTGTGGAAGATGACTTCGCAGGCGTGGTTCGCGATGACGTCACCCTGTTTATTTCCGCAGTTCAGGATCAAGTCGTGCCGGACAACACGCTGGCTTGGGTGTGGGTTCGTGGCCTGGATGAACTGTATGCGGAATGGAGCGAAGTTGTCTCTACCAATTTCCGTGACTCGAGCGGTCCGGCCATGACGGAAATCGGCGAACAGCCGTGGGGTCGCGAATTTGCTCTGCGTGACCCGGCTGGGAACTGTGTCCATTTCGTGGCTGAAGAACAAGATTGAGTTGAGATGACACTGTGATCTAAAAAGAGCGACTTCGGTCGCTCTTTTTTTTACCTGATAAAATGAAGTTAAAGGACTGCGTCATGATTAAGAAAATTTTTGCCCTTCCGGTCATCGAACAAATCTCCCCTGTCCTCTCCCGTCGTAAACTGGATGAACTGGACCTCATTGTGGTCGATCATCCGCAGGTAAAAGCCTCT gltA-DAS+4-ampRGTATTCCGTCTTCCATGTTCACCGTCATTTTCGCAATGGCACGTACCGTTGGC 62TGGATCGCCCACTGGAGCGAAATGCACAGTGACGGTATGAAGATTGCCCGTCCGCGTCAGCTGTATACAGGATATGAAAAACGCGACTTTAAAAGCGATATCAAGCGTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCTTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTACTTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACTAATGGTTGATTGCTAAGTTGTAAATATTTTAACCCGCCGTTCATATGGCGGGTTGATTTTTATATGCCTAAACACAAAAAATTGTAAAAATAAAATCCATTAACAGACCTATATAGATATTTAAAAAGAATAGAACAGCTCAAATTATCAGCAACCCAATACTTTCAATTAAAAACTTCATGGTAGTCGCATTTATAACCCTATGAAA gltA-DAS+4-purRACCGTCATTTTCGCAATGGCACGTACCGTTGGCTGGATCGCCCACTGGAGCG 63AAATGCACAGTGACGGTATGAAGATTGCCCGTCCGCGTCAGCTGTATACAGGATATGAAAAACGCGACTTTAAAAGCGATATCAAGCGTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGACTGAATACAAGCCCACGGTACGCTTGGCGACGCGCGACGATGTTCCCCGCGCTGTTCGTACATTAGCTGCGGCCTTTGCAGATTACCCAGCGACGCGCCATACGGTCGATCCGGACCGCCATATCGAGCGTGTCACAGAATTGCAGGAACTTTTCTTAACTCGCGTGGGCCTTGACATCGGAAAGGTCTGGGTGGCTGACGATGGCGCTGCACTGGCTGTTTGGACCACTCCGGAGAGTGTAGAGGCTGGTGCAGTGTTCGCCGAAATTGGTCCTCGTATGGCCGAATTAAGTGGAAGTCGTCTGGCAGCCCAACAACAAATGGAAGGGTTGCTTGCGCCCCACCGTCCGAAAGAACCCGCGTGGTTCCTTGCCACCGTTGGAGTAAGCCCAGATCACCAGGGGAAGGGTTTAGGATCTGCCGTAGTTTTACCAGGTGTGGAGGCAGCAGAACGTGCGGGAGTTCCGGCCTTCCTTGAGACGTCGGCGCCGCGCAATTTACCGTTTTACGAACGTCTTGGATTCACCGTTACGGCGGACGTGGAGGTGCCGGAGGGACCCCGTACTTGGTGTATGACTCGTAAACCGGGAGCCTGATAATGGTTGATTGCTAAGTTGTAAATATTTTAACCCGCCGTTCATATGGCGGGTTGATTTTTATATGCCTAAACACAAAAAATTGTAAAAATAAAATCCATTAACAGACCTATATAGATATTTAAAAAGAATAGAACAGCTCAAATTATCAGCAACCCA gltA-DAS+4-zeoRGTATTCCGTCTTCCATGTTCACCGTCATTTTCGCAATGGCACGTACCGTTGGC 64TGGATCGCCCACTGGAGCGAAATGCACAGTGACGGTATGAAGATTGCCCGTCCGCGTCAGCTGTATACAGGATATGAAAAACGCGACTTTAAAAGCGATATCAAGCGTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCATCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGGACTGAGGATAAGTAATGGTTGATTGCTAAGTTGTAAATATTTTAACCCGCCGTTCATATGGCGGGTTGATTTTTATATGCCTAAACACAAAAAATTGTAAAAATAAAATCCATTAACAGACCTATATAGATATTTAAAAAGAATAGAACAGCTCAAATTATCAGCAACCCAATACTTTCAATTAAAAACTTCATGGTAGTCGCATTTATAACCCTATGAAA lpd-DAS+4-gentRGCGGCGAGCTGCTGGGTGAAATCGGCCTGGCAATCGAAATGGGTTGTGATG 65CTGAAGACATCGCACTGACCATCCACGCGCACCCGACTCTGCACGAGTCTGTGGGCCTGGCGGCAGAAGTGTTCGAAGGTAGCATTACCGACCTGCCGAACCCGAAAGCGAAGAAGAAGGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGCGAATCCATGTGGGAGTTTATTCTTGACACAGATATTTATGATATAATAACTGAGTAAGCTTAACATAAGGAGGAAAAACATATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAATTTTTCGTTTGCCGGAACATCCGGCAATTAAAAAAGCGGCTAACCACGCCGCTTTTTTTACGTCTGCAATTTACCTTTCCAGTCTTCTTGCTCCACGTTCAGAGAGACGTTCGCATACTGCTGACCGTTGCTCGTTATTCAGCCT GACAGTATGGTTACTGTCudhA-DAS+4-bsdR TCTGGGTATTCACTGCTTTGGCGAGCGCGCTGCCGAAATTATTCATATCGGT 66CAGGCGATTATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTCGTCAACACCACCTTTAACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCGTTAAACGGTTTAAACCGCCTGTTTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCACTGAGAAGATCACCATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCCTACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTCTGGCTGTCAGCCACCCCTACTCTGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAATGGCAAGCTGGTCAAAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAAGTAAAACTTTATCGAAATGGCCATCCATTCTTGCGCGGATGGCCTCTGCCAGCTGCTCATAGCGGCTGCGCAGCGGTGAGCCAGGACGATAAACCAGGCCAATAGTGCGGCGTGGTTCCGGCTTAATGCACGG zwf-DAS+4-bsdRGAAGTGGAAGAAGCCTGGAAATGGGTAGACTCCATTACTGAGGCGTGGGCG 67ATGGACAATGATGCGCCGAAACCGTATCAGGCCGGAACCTGGGGACCCGTTGCCTCGGTGGCGATGATTACCCGTGATGGTCGTTCCTGGAATGAGTTTGAGGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCACTGAGAACATCACCATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCCTACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGTCAGCCACCCCTACTCTGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATCTGCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAATGGCAAGCTGGTCAAAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAAGTAATATCTGCGCTTATCCTTTATGGTTATTTTACCGGTAACATGATCTTGCGCAGATTGTAGAACAATTTTTACACTTTCAGGCCTCGTGCGGATTCACCCACGAGGCTTTTTTTATTACACTGACTGAAACGTTTTTGCCCTATGAGCTCCGGTTACAGGCGTTTCAGTCATAAATCCTCTGAATGAAACGCGTTGTGAATC dadX-DAS+4-purRGCGTGCGCACCATGACGGTGGGGACCGTCTCGATGGATATGCTAGCGGTCG 68ATTTAACGCCTTGCCCGCAGGCGGGTATTGGTACGCCGGTTGAGCTGTGGGGCAAGGAGATCAAAATTGATGATGTCGCCGCCGCTGCCGGAACGGTGGGCTATGAGTTGATGTGCGCGCTGGCGCTACGCGTCCCGGTTGTGACGGTGGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGACTGAATACAAGCCCACGGTACGCTTGGCGACGCGCGACGATGTTCCCCGCGCTGTTCGTACATTAGCTGCGGCCTTTGCAGATTACCCAGCGACGCGCCATACGGTCGATCCGGACCGCCATATCGACCGTCTCACAGAATTGCAGGAACTTTTCTTAACTCGCGTGGGCCTTGACATCGGAAAGGTCTGGGTGGCTGACGATGGCGCTGCAGTGGCTGTTTGGACCACTCCGGAGAGTGTAGAGGCTGGTGCAGTGTTCGCCGAAATTGGTCCTCGTATGGCCGAATTAAGTGGAAGTCGTCTGGCAGCCCAACAACAAATGGAAGGGTTGCTTGCGCCCCACCGTCCGAAAGAACCCGCGTGGTTCCTTGCCACCGTTGGAGTAAGCCCAGATCACCAGGGGAAGGGTTTAGGATCTGCCGTAGTTTTACCAGGTGTGGAGGCAGCAGAACGTGCGGGAGTTCCGGCCTTCCTTGAGACGTCGGCGCCGCGCAATTTACCGTTTTACGAACGTCTTGGATTCACCGTTACGGCGGACGTGGAGGTGCCGGAGGGACCCCGTACTTGGTGTATGACTCGTAAACCGGGAGCCTGATAACTTGTTGTAAGCCGGATCGGAGGCAACGTCTTCTGGGTGCAAAAAAATCATCCATCCGGGTGGTCAGCAACTGTAGTTGTTAATGTGACAGAGCCATTGCCCATGATAGTGTCCATTAAAAGGATGGACACTATTTCCCCGGAACCTGAACTCACCGCACAGGCGTTCTACATAAAACGCTTACGCTTCATTGTTGACTC

Section 4: Dynamics Control Over Protein Levels.

Plasmids expressing fluorescent proteins and silencing guides weretransformed into the corresponding hosts strain listed in Table 8.Strains were evaluated in triplicate in an m2p-labs Biolector™, whichsimultaneously measures fluorescence including GiFPuv and mCherrylevels, as well as biomass levels.

TABLE 8 Strains used for Dynamic Control over protein levels SyntheticMetabolic Microbe Valves Plasmid Host Strain E. coli RFP-control pCDF-DLF_0002  mcherry1 + PSMART- IN:yibDp-GFPuv Proteolysis pCDF- DLF_0025 mcherry2 + PSMART- IN:yibDp-GFPuv Silencing pCDF- DLF_01517 mcherry1 +pCASCADE- proD + pSMART- IN:yibDp-GFPuv Proteolysis + pCDF- DLF_0025 Silencing mcherry2 + pCASCADE- proD + pSMART- IN:yibDp-GFPuv

OD600 readings were corrected using the formula below, where OD600refers to an offline measurement. OD600* refers to Biolector biomassreading, t0 indicates the start point, and if indicates the final point.

$\begin{matrix}{{{OD}\; 600_{t}} = {{\left( {{{OD}\; 600_{t}^{*}} - {{OD}\; 600_{t\; 0}^{*}}} \right)*\frac{\left( {{{OD}\; 600_{tf}} - {{OD}\; 600_{t\; 0}}} \right)}{\left( {{{OD}\; 600_{tf}^{*}} - {{OD}\; 600_{t\; 0}^{*}}} \right)}} + 0.25}} & {{Equation}\mspace{14mu} S\; 1}\end{matrix}$

Section 5: Metabolic Control

Near Equilibrium Reactions

The impact of Valves on metabolite pools for near equilibrium reactionsis illustrated using the G6P node as an example. Abbreviations: Gluc,glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; 6PGI,6-phosphate-gluconolactone.

G6P Node without Valves

Steady State Mass balance J ₁ =J ₂ +J ₃  Equation S2:

$\begin{matrix}{{{Net}\mspace{14mu}{Flux}} = {J_{i} = {e^{\frac{- {dG}}{RT}} - 1}}} & {{Equation}\mspace{14mu}{S3}} \\{{e^{\frac{{- {dG}}\; 1}{RT}} - 1} = {e^{\frac{{- {dG}}\; 2}{RT}} - 1 + e^{\frac{{- {dG}}\; 3}{RT}} - 1}} & {{Equation}\mspace{14mu}{S4}} \\{e^{\frac{{- {dG}}\; 1}{RT}} = {e^{\frac{{- {dG}}\; 2}{RT}} + e^{\frac{{- {dG}}\; 3}{RT}} - 1}} & {{Equation}\mspace{14mu}{S5}} \\{{{Keq}\; 1} = {{{Keq}\; 2} + {{Keq}\; 3} - 1}} & {{Equation}\mspace{14mu}{S6}} \\{{{{Keq}\; 1} + 1} = {{{Keq}\; 2} + {{Keq}\; 3}}} & {{Equation}\mspace{14mu}{S7}} \\{{\frac{\left\lbrack {G\; 6P} \right\rbrack}{\lbrack{Gluc}\rbrack} + 1} = {\frac{\left\lbrack {F\; 6P} \right\rbrack}{\left\lbrack {G\; 6P} \right\rbrack} + \frac{\left\lbrack {6{PGl}} \right\rbrack}{\left\lbrack {G\; 6P} \right\rbrack}}} & {{Equation}\mspace{14mu}{S8}} \\{{\frac{\left\lbrack {G\; 6P} \right\rbrack}{\lbrack{Gluc}\rbrack} + 1} = \frac{\left\lbrack {F\; 6P} \right\rbrack + \left\lbrack {6{PGl}} \right\rbrack}{\left\lbrack {G\; 6P} \right\rbrack}} & {{Equation}\mspace{14mu}{S9}} \\{{\frac{\left\lbrack {G\; 6P} \right\rbrack^{2}}{\lbrack{Gluc}\rbrack} + \left\lbrack {G\; 6P} \right\rbrack} = {\left\lbrack {F\; 6P} \right\rbrack + \left\lbrack {6{PGl}} \right\rbrack}} & {{Equation}\mspace{14mu}{S10}} \\{\left\lbrack {F\; 6P} \right\rbrack = {\frac{\left\lbrack {G\; 6P} \right\rbrack^{2}}{\lbrack{Gluc}\rbrack} + \left\lbrack {G\; 6P} \right\rbrack - \left\lbrack {6{PGl}} \right\rbrack}} & {{Equation}\mspace{14mu} S\; 11}\end{matrix}$

G6P Node with Valves

When zwf valve is in effect, J₃≈0.

Steady State Mass balance J ₁ =J ₂  Equation S12:

$\begin{matrix}{{{Net}\mspace{14mu}{Flux}} = {J_{i} = {e^{\frac{- {dG}}{RT}} - 1}}} & {{Equation}\mspace{14mu}{S13}} \\{{e^{\frac{{- {dG}}\; 1}{RT}} - 1} = {e^{\frac{{- {dG}}\; 2}{RT}} - 1}} & {{Equation}\mspace{14mu}{S14}} \\{{{Keq}\; 1} = {{Keq}\; 2}} & {{Equation}\mspace{14mu}{S15}} \\{\frac{\left\lbrack {G\; 6P} \right\rbrack}{\lbrack{Gluc}\rbrack} = \frac{\left\lbrack {F\; 6P} \right\rbrack}{\left\lbrack {G\; 6P} \right\rbrack}} & {{Equation}\mspace{14mu}{S16}} \\{\left\lbrack {F\; 6P} \right\rbrack = \frac{\left\lbrack {G\; 6P} \right\rbrack^{2}}{\lbrack{Gluc}\rbrack}} & {{Equation}\mspace{14mu}{S17}}\end{matrix}$

Impact of Valves

$\begin{matrix}{{\left\lbrack {F\; 6P} \right\rbrack\mspace{11mu}{network}} = {\frac{\left\lbrack {G\; 6P} \right\rbrack^{2}}{\lbrack{Gluc}\rbrack} + \left\lbrack {G\; 6P} \right\rbrack - \left\lbrack {6{PGl}} \right\rbrack}} & {{Equation}\mspace{14mu}{S11}} \\{{\left\lbrack {F\; 6P} \right\rbrack\mspace{11mu}{value}} = \frac{\left\lbrack {G\; 6P} \right\rbrack^{2}}{\lbrack{Gluc}\rbrack}} & {{Equation}\mspace{14mu} S\; 17}\end{matrix}$

-   -   Since close to equilibrium [6PGI]>[G6P]

[F6P]valve>=[F6P]network

-   -   The removal of thermodynamically favored reactions near        equilibrium from the network will result in increased metabolite        pools.

Section 6: Gene Silencing Arrays & Pathway Expression Constructs

The design and construction of CASCADE guides and guide arrays isillustrated below in FIG. 14 and FIG. 15A-B. The pCASCADE-controlplasmid was prepared by swapping the pTet promoter in pcrRNA.Tet⁸⁸ withan insulated low phosphate induced ugpB promoter⁸². Two promoters wereresponsible for regulating gltA gene, and sgRNA was designed for bothpromoters, resulting in guide gltA1 (G1) and gltA2 (G2).⁸⁹ Fourpromoters were responsible for regulating gapA gene, and sgRNA wasdesigned for the first promoter, since during exponential phase ofgrowth, gapA mRNAs were mainly initiated at the highly efficient gapA P1promoter and remained high during stationary phase compared to the otherthree gapA promoters.⁹⁰ Multiple promoters upstream of lpd gene w % ereinvolved in lpd regulation(https://ecocyc.org/gene?orgid=ECOLI&id=EG10543#tab=showAll), thusdesign of unique and effective sgRNA for lpd only was not possible.Promoter sequences for fabI, udhA and zwf were obtained from EcoCycdatabase (https://ecocyc.org/). To design CASCADE guide array, CASCADEPAM sites near the −35 or −10 box of the promoter of interest wereidentified, 30 bp at the 3′ end of PAM site was selected as the guidesequence and cloned into pCASCADE plasmid using Q5 site-directedmutagenesis (NEB, MA) following manufacturer's protocol, with themodification that 5% v/v DMSO was added to the Q5 PCR reaction. ThepCASCADE-control vector was used as template, pCASCADE plasmids witharrays of two or more guides were prepared as illustrated in FIG. 15A-B.The pCASCADE guide array plasmid was prepared by sequentially amplifyingcomplementary halves of each smaller guide plasmid by PCR, followed bysubsequent DNA assembly. Table 9 lists sgRNA guide sequences and primersused to construct them. All pCASCADE silencing plasmids are listed inTable 10 below and are available at Addgene.

TABLE 9List of sgRNA guide sequences and primers used to construct them. Spacersare italicized. sgRNA/Primer Name Sequence SEQ ID NO Template fabITCGAGTTCCCCGCGCCAGCGGG  69 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCAGCGGGGATAA ACCG fabI-FOR GTTTATCTGTTCGTATCGAGTT  70pCASCADE control CCCCGCGCCAGCGGGGATAAAC CGAAAAAAAAACCCC fabI-REVGGTTATTATAATCACGGTTTA  71 TCCCCGCTGGCGCGGGGAACT CGAGGTGGTACCAGAT gapAP1TCGAGTTCCCCGCGCCAGCGGG  72 GATAAACCGGTTTTTGTAATTTTACAGGCAACCTTTTATTCGAGT TCCCCGCGCCAGCGGGGATAAA CCG gapAP1-FORCAGGCAACCTTTTATTCGAGTT  73 pCASCADE control CCCCGCGCCAGCGGGGATAAACCGAAAAAAAAACCCC gapAP1-REV TAAAATTACAAAAACCGGTTT  74ATCCCCGCTGGCGCGGGGAAC TCGAGGTGGTACCAGATC gltA1 TCGAGTTCCCCGCGCCAGCGGG 75 GATAAACCGAAAAGCATATAAT GCGTAAAAGTTATGAAGTTCG AGTTCCCCGCGCCAGCGGGGATAAACCG gltA1-FOR GCGTAAAAGTTATGAAGTTCG  76 pCASACADE controlAGTTCCCCGCGCCAGCGGGGAT AAACCGAAAAAAAAACCCCC gltA1-REVATTATATGCTTTTCGGTTTATC  77 CCCGCTGGCGCGGGGAACTCG AGGTGGTACCAGATCT gltA2TCGAGTTCCCCGCGCCAGCGGG  78 GATAAACCGTATTGACCAATTC ATTCGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGAT AAACCG gltA2-FOR GGGACAGTTATTAGTTCGAGTT  79pCASCADE control CCCCGCGCCAGCGGGGATAAAC CGAAAAAAAAACCCC gltA2-REVGAATGAATTGGTCAATACGGT  80 TTATCCCCGCTGGCGCGGGGA ACTCGAGGTGGTACCAGATCTproD TCGAGTTCCCCGCGCCAGCGGG  81 GATAAACCGAGTGGTTCGCTGGATAACTTTACGGGCATGCTCGAG TTCCCCGCGCCGCGGGGATAA ACCG proD-FORAACTTTACGGGCATGCTCGAGT  82 pCASCADE control TCCCCGCGCCAGCGGGGATAAACCGAAAAAAAAACCCC proD-REV ATCCAGCAACCACTCGGTTTAT  83CCCCGCTGGCGCGGGGAACTC GAGGTGGTACCAGATCT udhA TCGAGTTCCCCGCGCAGCGGG  84GATAAACCGTTACCATTCTGTT GCTTTTATGTATAAGAATCGAG TTCCCCGCGCCAGCGGGGATAAACCG udhA-FOR TTTTATGTATAAGAATCGAGTT  85 pCASCADE controlCCCCGCGCCAGCGGGGATAAAC CGAAAAAAAAACCCC udhA-REV GCAACAGAATGGTAACGGTTT 86 ATCCCCGCTGGCGCGGGGAAC TCGAGGTGGTACCAGATC zwf TCGAGTTCCCCGCGCCAGCGGG 87 GATAAACCGCTCGTAAAAGCAG TACAGTGCACCGTAAGATCGA GTTCCCCGCGCCAGCGGGGATAAACCG zwf-FOR CAGTGCACCGTAAGATCGAGTT  88 control CCCCGCGCCAGCGGGGATAAACCGAAAAAAAAACCCC zwf-REV TACTGCTTTTACGAGCGGTTTA  89 TCCCCGCTGGCGCGGGGAACTCGAGGTGGTACCAGATC FG1 TCGAGTTCCCCGCGCCAGCGGG  90 GATAAACCGTTGATTATAATAACCGTTTATCTGTTCGTATCGAG TTCCCCGCGCCAGCGGGGATAA ACCGAAAAGCATATAATGCGTAAAAGTTATGAAGTTCGAGTTC CCCGCGCCAGCGGGGATAAACC G gltA-FORGCGCCAGCGGGGATAAACCGA  91 pCASCADE-gltA1 AAAGCATATAATGCG pCASCADE-REVCTTGCCCGCCTGATGAATGCTC  92 ATCCGG pCASCADE-FOR CCGGATGAGCATTCATCAGGC  93pCASCADE-fabI GGGCAAG fabI-REV CGGTTTATCCCCGCTGGCGCG  94GGGAACTCGATACGAACAGAT AAACGGTTATTATAATC FG2 TCGAGTTCCCCGCGCCAGCGGG  95GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAG TTCCCCGCGCCAGCGGGGATAAACCGTATTGACCAATTCATTCG GGACAGTTATTAGTTCGAGTTC CCCGCGCCAGCGGGGATAAACC GgltA2-FOR GCGCCAGCGGGGATAAACCGT  96 pCASCADE-gltA2 ATTGACCAATTCATTCpCASCADE-REV CTTGCCCGCCTGATGAATGCTC  97 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC  98 pCASCADE-fabI GGGCAAG fabI-REVCGGTTTATCCCCGCTGGCGCG  99 GGGAACTCGATACGAACAGAT AAACGGTTATTATAATC FUTCGAGTTCCCCGCGCCAGCGGG 100 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCAGCGGGGATAA ACCGTTACCATTCTGTTGCTTT TATGTATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCG udhA-FOR GCGCCAGCGGGGATAAACCGT 101 pCASCADE-udhATACCATTCTGTTG pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 102 ATCCGGpCASCADE-FOR CCGGATGAGCATTCATCAGGC 103 pCASCADE-fabI GGGCAAG fabI-REVCGGTTTATCCCCGCTGGCGCG 104 GGGAACTCGATACGAACAGAT AAACGGTTATTATAATC FZTCGAGTTCCCCGCGCCAGCGGG 105 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCAGCGGGGATAA ACCGCTCGTAAAAGCAGTACA GTGCACCGTAAGATCGAGTTCCCCGCGCCAGCGGGGATAAACCG zwf-FOR GCGCCAGCGGGGATAAACCGC 106 pCASCADE-zwfTCGTAAAAG pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 107 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC 108 pCASCADE-fabI GGGCAAG fabI-REVCGGTTTATCCCCGCTGGCGCG 109 GGGAACTCGATACGAACAGAT AAACGGTTATTATAATC G1G2TCGAGTTCCCCGCGCCAGCGGG 110 GATAAACCGAAAAGCATATAAT GCGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGAT AAACCGTATTGACCAATTCATT CGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAA CCG gltA2-FOR GCGCCAGCGGGGATAAACCGT 111pCASCADE-gltA2 ATTGACCAATTCATTC pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 112ATCCGG CCGGATGAGCATTCATCAGGC 113 pCASCADE-gltA1 GGGCAAG gltA1-REVCGGTTTATCCCCGCTGGCGCG 114 GGGAACTCGAACTTCATAACT TTTAC G1UTCGAGTTCCCCGCGCCAGCGGG 115 GATAAACCGAAAAGCATATAATG CGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGATA AACCGTTACCATTCTGTTGCTT TTATGTATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCG udhA-FOR GCGCCAGCGGGGATAAACCGT 116 pCASCADE-udhATACCATTCTGTTG pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 117 ATCCGGpCASCADE-FOR CCGGATGAGCATTCATCAGGC 118 pCASCADE-gltA1 GGGCAAG gltA1-REVCGGTTTATCCCCGCTGGCGCG 119 GGGAACTCGAACTTCATAACT TTTAC G1ZTCGAGTTCCCCGCGCCAGCGGG 120 GATAAACCGAAAAGCATATAAT GCGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGAT AAACCGCTCGTAAAAGCAGTA CAGTGCACCGTAAGATCGAGTTCCCCGCGCCAGCGGGGATAAAC CG zwf-FOR GCGCCAGCGGGGATAAACCGC 121 pCASCADE-zwfTCGTAAAAG pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 122 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC 123 pCASCADE-gltA1 GGGCAAG gltA1-REVCGGTTTATCCCCGCTGGCGCG 124 GGGAACTCGAACTTCATAACT TTTAC G2UTCGAGTTCCCCGCGCCAGCGGG 125 GATAAACCGTATTGACCAATTCATTCGGGACAGTTATTAGTTCGA GTTCCCCGCGCCAGCGGGGATA AACCGTTACCATTCTGTTGCTTTTATGTATAAGAATCGAGTTCC CCGCGCCAGCGGGGATAAACCG udhA-FORGCGCCAGCGGGGATAAACCGT 126 pCASCADE-udhA TACCATTCTGTTG pCASCADE-REVCTTGCCCGCCTGATGAATGCTC 127 ATCCGG pCASCADE-FOR CCGGATGAGCATTCATCAGGC 128pCASCADE-gltA2 GGGCAAG gltA2-REV CGGTTTATCCCCGCTGGCGCG 129GGGAACTCGAACTAATAACTG TC G2Z TCGAGTTCCCCGCGCCAGCGGG 130GATAAACCGTATTGACCAATTCA TTCGGGACAGTTATTAGTTCGA GTTCCCCGCGCCAGCGGGGATAAACCGCTCGTAAAAGCAGTAC AGTGCACCGTAAGATCGAGTTC CCCGCGCCAGCGGGGATAAACC Gzwf-FOR GCGCCAGCGGGGATAAACCGC 131 pCASCADE-zwf TCGTAAAAG pCASCADE-REVCTTGCCCGCCTGATGAATGCTC 132 ATCCGG pCASCADE-FOR CCGGATGAGCATTCATCAGGC 133pCASCADE-gltA2 GGGCAAG gltA2-REV CGGTTTATCCCCGCTGGCGCG 134GGGAACTCGAACTAATAACTG TC UZ TCGAGTTCCCCGCGCCAGCGGG 135GATAAACCGTTACCATTCTGTT GCCTTTTATGTATAAGAATCGAG TTCCCCGCGCCAGCGGGGATAAACCGCTCGTAAAAGCAGTACA GTGCACCGTAAGATCGAGTTCC CCGCGCCAGCGGGGATAAACCGzwf-FOR GCGCCAGCGGGGATAAACCGC 136 pCASCADE-zwf TCGTAAAAG pCASCADE-REVCTTGCCCGCCTGATGAATGCTC 137 ATCCGG pCASCADE-FOR CCGGATGAGCATTCATCAGGC 138pCASCADE-udhA GGGCAAG udhA-REV CGGTTTATCCCCGCTGGCGCG 139GGGAACTCGATTCTTATACAT AAAAGC FG1G2 TCGAGTTCCCCGCGCCAGCGGG 140GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAG TTCCCCGCGCCAGCGGGGATAAACCGAAAAGCATATAATGCGT AAAAGTTATGAAGTTCGAGTTC CCCGCGCCAGCGGGGATAAACCGTATTGACCAATTCATTCGGG ACAGTTATTAGTTCGAGTTCCC CGCGCCAGCGGGGATAAACCGgltA2-FOR GCGCCAGCGGGGATAAACCGT 141 pCASCADE-gltA2 ATTGACCAATTCATTCpCASCADE-REV CTTGCCCGCCTGATGAATGCTC 142 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC 143 pCASCADE-FG1 GGGCAAG gltA1-REVCGGTTTATCCCCGCTGGCGCG 144 GGGAACTCGAACTTCATAACT TTTAC G1G2ATCGAGTTCCCCGCGCCAGCGGG 145 GATAAACCGAAAAGCATATAAT GCGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGAT AAACCGTATTGACCAATTCATT CGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAA CCGGTTTTTGTAATTTTACAGG CAACCTTTTATTCGAGTTCCCCGCGCCAGCGGGGATAAACCG gapAP1-FOR GCGCCAGCGGGGATAAACCGG 146pCASCADE-gpaAP1 TTTTTGTAATTTTACAGGC pCASCADE-REV CTTGCCCGCCTGATGAATGCTC147 ATCCGG pCASCADE-FOR CCGGATGAGCATTCATCAGGC 148 pCASCADE-G1G2 GGGCAAGgltA2-REV CGGTTTATCCCCGCTGGCGCG 149 GGGAACTCGAACTAATAACTG TC G1G2UTCGAGTTCCCCGCGCCAGCGGG 150 GATAAACCGAAAAGCATATAAT GCGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGAT AAACCGTATTGACCAATTCATT CGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAA CCGTTACCATTCTGTTGCTTTT ATGTATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCG udhA-FOR GCGCCAGCGGGGATAAACCGT 151 pCASCADE-udhATACCATTCTGTTG pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 152 ATCCGGpCASCADE-FOR CCGGATGAGCATTCATCAGGC 153 pCASCADE-G1G2 GGGCAAG gltA2-REVCGGTTTATCCCCGCTGGCGCG 154 GGGAACTCGAACTAATAACTG TC G1G2ZTCGAGTTCCCCGCGCCAGCGGG 155 GATAAACCGAAAAGCATATAAT GCGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGAT AAACCGTATTGACCAATTCATT CGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAA CCGCTCGTAAAAGCAGTACAG TGCACCGTAAGATCGAGTTCCCCGCGCCAGCGGGGATAAACCG zwf-FOR GCGCCAGCGGGGATAAACCGC 156 pCASCADE-zwfTCGTAAAAG pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 157 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC 158 pCASCADE-G1G2 GGGCAAG gltA2-REVCGGTTTATCCCCGCTGGCGCG 159 GGGAACTCGAACTAATAACTG TC FG1G2ATCGAGTTCCCCGCGCCAGCGGG 160 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCAGCGGGGATAA ACCGAAAAGCATATAATGCGT AAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACC GTATTGACCAATTCATTCGGG ACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACCGG TTTTTGTAATTTTACAGGCAAC CTTTTATTCGAGTTCCCCGCGCCAGCGGGGATAAACCG gapAP1-FOR GCGCCAGCGGGGATAAACCGG 161 pCASCADE-gapAP1TTTTTGTAATTTTACAGGC pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 162 ATCCGGpCASCADE-FOR CCGGATGAGCATTCATCAGGC 163 pCASCADE-FG1G2 GGGCAAG gltA2-REVCGGTTTATCCCCGCTGGCGCG 164 GGGAACTCGAACTAATAACTG TC FG1G2UTCGAGTTCCCCGCGCCAGCGGG 165 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCAGCGGGGATAA ACCGAAAAGCATATAATGCGT AAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACC GTATTGACCAATTCATTCGGG ACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACCGT TACCATTCTGTTGCTTTTATGT ATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCG gltA2-FOR GCGCCAGCGGGGATAAACCGT 166 pCASCADE-udhAATTGACCAATTCATTC pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 167 ATCCGGpCASCADE-FOR CCGGATGAGCATTCATCAGGC 168 pCASCADE-FG1G2 GGGCAAG gltA1-REVCGGTTTATCCCCGCTGGCGCG 169 GGGAACTCGAACTTCATAACT TTTAC FG1G2ZTCGAGTTCCCCGCGCCAGCGGG 170 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCAGCGGGGATAA ACCGAAAAGCATATAATGCGT AAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACC GTATTGACCAATTCATTCGGG ACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACCGC TCGTAAAAGCAGTACAGTGCA CCGTAAGATCGAGTTCCCCGCGCCAGCGGGGATAAACCG gltA2-FOR GCGCCAGCGGGGATAAACCGT 171 pCASCADE-zwfATTGACCAATTCATTC pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 172 ATCCGGpCASCADE-FOR CCGGATGAGCATTCATCAGGC 173 pCASCADE-FG1G2 GGGCAAG gltA1-REVCGGTTTATCCCCGCTGGCGCG 174 GGGAACTCGAACTTCATAACT TTTAC G1G2UATCGAGTTCCCCGCGCCAGCGGG 175 GATAAACCGAAAAGCATATAAT GCGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGAT AAACCGTATTGACCAATTCATT CGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAA CCGTTACCATTCTGTTGCTTTT ATGTATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCGGT TTTTGTAATTTTACAGGCAAC CTTTTATTCGAGTTCCCCGCGCCAGCGGGGATAAACCG gapA1-FOR GCGCCAGCGGGGATAAACCGG 176 pCASCADE-AP1TTTTTGTAATTTTACAGGC pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 177 ATCCGGpCASCADE-FOR CCGGATGAGCATTCATCAGGC 178 pCASCADE-G1G2U GGGCAAG udhA-REVCGGTTTATCCCCGCTGGCGCG 179 GGGAACTCGATTCTTATACAT AAAAGC G1G2UZTCGAGTTCCCCGCGCCAGCGGG 180 GATAAACCGAAAAGCATATAAT GCGTAAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGAT AAACCGTATTGACCAATTCATT CGGGACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAA CCGTTACCATTCTGTTGCTTTT ATGTATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCGCT CGTAAAAGCAGTACAGTGCAC CGTAAGATCGAGTTCCCCGCGCCAGCGGGGATAAACCG zwf-FOR GCGCCAGCGGGGATAAACCGC 181 pCASCADE-zwfTCGTAAAAG pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 182 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC 183 pCASCADE-G1G2U GGGCAAG udhA-REVCGGTTTATCCCCGCTGGCGCG 184 GGGAACTCGATTCTTATACAT AAAAGC FG1G2UATCGAGTTCCCCGCGCCAGCGGG 185 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCCAGCGGGGATAA ACCGAAAAGCATATAATGCGT AAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACC GTATTGACCAATTCATTCGGG ACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACCGT TACCATTCTGTTGCTTTTATGT ATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCGGTTTTT GTAATTTTACAGGCAACCTTT TATTCGAGTTCCCCGCGCCAGCGGGGATAAACCG gapAP1-FOR GCGCCAGCGGGGATAAACCGG 186 pCASCADE-gpaAP1TTTTTGTAATTTTACAGGC pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 187 ATCCGGpCASCADE-FOR CCGGATGAGCATTCATCAGGC 188 pCASCADE- GGGCAAG FG1G2U udhA-REVCGGTTTATCCCCGCTGGCGCG 189 GGGAACTCGATTCTTATACAT AAAAGC FG1G2UZTCGAGTTCCCCGCGCCAGCGGG 190 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCAGCGGGGATAA ACCGAAAAGCATATAATGCGT AAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACC GTATTGACCAATTCATTCGGG ACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACCGT TACCATTCTGTTGCTTTTATGT ATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCGCTCGTA AAAGCAGTACAGTGCACCGTA AGATCGAGTTCCCCGCGCCAGCGGGGATAAACCG zwf-FOR GCGCCAGCGGGGATAAACCGC 191 pCASCADE-zwf TCGTAAAAGpCASCADE-REV CTTGCCCGCCTGATGAATGCTC 192 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC 193 pCASCADE- GGGCAAG FG1G2U udhA-REVCGGTTTATCCCCGCTGGCGCG 194 GGGAACTCGATTCTTATACAT AAAAGC FG1G2UZATCGAGTTCCCCGCGCCAGCGGG 195 GATAAACCGTTGATTATAATAA CCGTTTATCTGTTCGTATCGAGTTCCCCGCGCCCAGCGGGGATAA ACCGAAAAGCATATAATGCGT AAAAGTTATGAAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACC GTATTGACCAATTCATTCGGG ACAGTTATTAGTTCGAGTTCCCCGCGCCAGCGGGGATAAACCGT TACCATTCTGTTGCTTTTATGT ATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAAACCGCTCGTA AGATCGAGTTCCCCGCGCCAGC GGGGATAAACCGGTTTTTGTAATTTTACAGGCAACCTTTTATTC GAGTTCCCCGCGCCCAGCGGGGA TAAACCG gapAP1-FORGCGCCAGCGGGGATAAACCGG 196 pCASCADE-gapA1 TTTTTGTAATTTTACAGGCpCASCADE-REV CTTGCCCGCCTGATGAATGCTC 197 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC 198 pCASCADE- GGGCAAG FG1G2UZ zwf-REVCGGTTTATCCCCGCTGGCGCG 199 GGGAACTCGATCTTACGGTGC ACTGTC UZTCGAGTTCCCCGCGCCAGCGGG 200 GATAAACCGTTACCATTCTGTT GCTTTTATGTATAAGAATCGAGTTCCCCGCGCCAGCGGGGATAA ACCGCTCGTAAAAGCAGTACA GTGCACCGTAAGATCGAGTTCCCCGCGCCAGCGGGGATAAACCG zwf-FOR GCGCCAGCGGGGATAAACCGC 201 pCASCADE-zwfTCGTAAAAG pCASCADE-REV CTTGCCCGCCTGATGAATGCTC 202 ATCCGG pCASCADE-FORCCGGATGAGCATTCATCAGGC 203 pCASCADE-udhA GGGCAAG udhA-REVCGGTTTATCCCCGCTGGCGCG 204 GGGAACTCGATTCTTATACAT AAAAGC

TABLE 10 List of plasmids used in this study. Plasmid Utilized in thisStudy Plasmid Purpose Source pSIM5 Recombineering and StrainConstruction Court Lab⁵⁴ pCP20 FRT kanamycin cassette curing Court Lab⁵⁴pSMART-HC-Kan Backbone Vector Lucigen pcrRNA.Tet pCASCADE-controlbackbone Beisel Lab³⁴ Plasmid Constructed in this Study Piasmid PlasmidName Addgene ID pSMART-Ala2 pSMART-HCKanIN:yibDp-ald*  71326 pSMART-Ala3pSMART-HCKan-IN:phoBp-ald*  71327 pSMART-Ala4 pSMART-HCKan-IN:phoBp-ald* 71328 pSMART-Ala5 pSMART-HCKan-IN:mipAp-ald*  71329 pSMART-Ala11pSMART-HCKan-proA-ald*  87172 pSMART-Ala12 pSMART-HCKan-proC-ald*  87173pSMART-Ala13 pSMART-HCKan-proD-ald*  87174 pSMART-Ala14pSMART-HCKan-proB-ald* 101079 pSMART-Ala15 pSMART-HCKan-HCEp-ald* 101080pSMART-Mev2 pSMART-IN:yibDp1-mvaE-IN:phoBp2-mvaS(A110G)  66642pSMART-Mev3 pSMART-IN:yibDp1-mvaE-IN:mipAp2-mvaS(A110G) 102761pSMART-Mev4 pSMART-IN:yibDp1-mvaE-IN:phoHp2-mvaS(A110G) 102762pSMART-Mev5 pSMART-IN:mipAp1-mvaE-IN:yibDp2-mvaS(A110G) 102763pSMART-3HP pSMART-3HP-NADPH-rhtA  87143 pCDF-mcherry1 pCDF-proD-mcherry 87144 pCDF-mcherry2 pCDF-proD-mcherry-DAS4  87145 pSMART-GFPuvpSMART-IN:yibDp-GFPuv  65822 pSMART-GFPuv2 pSMART-IN:phoBp-GFPuv  71517pSMART-GFPuv3 pSMART-IN:phoUp-GFPuv  71518 pSMART-GFPuv4pSMART-IN:phoHp-GFPuv  71519 pSMART-GFPuv5 pSMART-IN:mipAp-GFPuv  71520pCASCADE-control pCASCADE  65821 pCASCADE-proD pCASCADE-proD  65820pCASCADE-gapAP1 pCASCADE-gapAP1  87146 pCASCADE-fabI pCASCADE-fabI 66635 pCASCADE-FG1 pCASCADE-fabI-gltA1  71340 pCASCADE-FG1G2pCASCADE-fabI-gltA1-gltA2  71342 pCASCADE-FG1G2ApCASCADE-fabI-gltA1-gltA2-gapA  87147 pCASCADE-FG1G2UpCASCADE-fabI-gltA1-gltA2-udhA  66637 pCASCADE-FG1G2UApCASCADE-fabI-gltA1-gltA2-udhA-gapA  87154 pCASCADE-FG1G2UZpCASCADE-fabI-gltA1-gltA2-udhA-zwf  87148 pCASCADE-FG1G2UZApCASCADE-fabI-gltA1-gltA2-udhA-zwf-gapA  87149 pCASCADE-FG1G2ZpCASCADE-fabI-gltA1-gltA2-zwf  66638 pCASCADE-FG2 pCASCADE-fabI-gltA2 71341 pCASCADE-FU pCASCADE-fabI-udhA  66636 pCASCADE-FZpCASCADE-fabI-zwf  71335 pCASCADE-G1G2 pCASCADE-gltA1-gltA2  71348pCASCADE-G1G2A pCASCADE-gltA1-gltA2-gapA  87150 pCASCADE-G1G2UpCASCADE-gltA1-gltA2-udhA  71343 pCASCADE-G1G2UApCASCADE-gltA1-gltA2-udhA-gapA  87151 pCASCADE-G1G2UZpCASCADE-gltA1-gltA2-udhA-zwf  87152 pCASCADE-G1G2ZpCASCADE-gltA1-gltA2-zwf  71347 pCASCADE-G1U pCASCADE-gltA1-udhA  71339pCASCADE-G1Z pCASCADE-gltA1-zwf  71337 pCASCADE-G2U pCASCADE-gltA2-udhA 65819 pCASCADE-G2Z pCASCADE-gltA2-zwf  71338 pCASCADE-gltA1pCASCADE-gltA1  71334 pCASCADE-gltA2 pCASCADE-gltA2  65817 pCASCADE-udhApCASCADE-udhA  65818 pCASCADE-UZ pCASCADE-udhA-zwf  87153 pCASCADE-zwfpCASCADE-zwf  65825

Section 7: 2-Stage Micro-Fermentations

E. coli Media Stock Solutions

-   -   10× concentrated Ammonium-Citrate 30 salts (1 L), mix 30 g of        (NH₄)₂SO₄ and 1.5 g citric acid in water with stirring, adjust        pH to 7.5 with 10 M NaOH. Autoclave and store at room        temperature (RT).    -   10× concentrated Ammonium-Citrate 90 salts (1 L), mix 90 g of        (NH₄)₂SO₄ and 2.5 g citric acid in water with stirring, adjust        pH to 7.5 with 10 M NaOH. Autoclave and store at RT.    -   1 M Potassium 3-(N-morpholino) propanesulfonic Acid (MOPS),        adjust to pH 7.4 with 50% KOH. Filter sterilize (0.2 μm) and        store at RT.    -   0.5 M potassium phosphate buffer, pH 6.8, mix 248.5 mL of 1.0 M        K₂HPO₃ and 251.5 mL of 1.0 M KH₂PO₄ and adjust to a final volume        of 1000 mL with ultrapure water. Filter sterilize (0.2 μm) and        store at RT.    -   2 M MgSO₄ and 10 mM CaSO₄ solutions. Filter sterilize (0.2 μm)        and store at RT.    -   50 g/L solution of thiamine-HCl. Filter sterilize (0.2 μm) and        store at 4° C.    -   500 g/L solution of glucose, dissolve by stirring with heat.        Cool, filter sterilize (0.2 μm), and store at RT.    -   100 g/L yeast extract, autoclave, and store at RT.    -   100 g/L casamino acid, autoclave, and store at RT.    -   500× Trace Metal Stock: Prepare a solution of micronutrients in        1000 mL of water containing 10 mL of concentrated H₂SO₄, 0.6 g        COSO₄.7H₂0, 5.0 g CuSO₄.5H₂O, 0.6 g ZnSO₄.7H₂O, 0.2 g        Na₂MoO₄.2H₂O, 0.1 g H₃BO₃, and 0.3 g MnSO₄.H₂O. Filter sterilize        (0.2 μm) and store at RT in the dark.    -   Prepare a fresh solution of 40 mM ferric sulfate heptahydrate in        water, filter sterilize (0.2 μm) before preparing media each        time.

Media Components

Prepare the final working medium by aseptically mixing stock solutionsbased on the following tables in the order written to minimizeprecipitation, then filter sterilize (with a 0.2 μm filter).

TABLE 11 Seed Media, pH 6.8: Ingredient Unit SM10 SM10++ (NH₄)₂SO₄ g/L 99 Citric Acid g/L 0.25 0.25 Potassium Phosphate mM 5 5 CoSO₄ · 7H₂O g/L0.0048 0.0048 CuSO₄ · 5H₂O g/L 0.04 0.04 ZnSO₄ · 7H₂O g/L 0.0048 0.0048Na₂MoO₄ · 2H₂O g/L 0.0016 0.0016 H₃BO₃ g/L 0.0008 0.0008 MnSO₄ · 7H₂Og/L 0.0024 0.0024 FeSO₄ · 7H₂O g/L 0.044 0.044 MgSO₄ mM 2.5 2.5 CaSO₄ mM0.06 0.06 Glucose g/L 45 45 MOPS niM 200 200 Thiamine-HCl g/L 001 0.01Yeast Extract g/L 1 2.5 Casarnino Acids g/L 0 2.5

TABLE 12 Production/Wash Media, pH 6.8: FGM3 No FGM3 FGM3 + 40 mMIngredient Unit FGM3 Phosphate Wash phosphate FGM10 (NH₄)₂SO₄ g/L 3 3 33 9 Citric Acid g/L 0.15 0.15 0.15 0.15 0.25 Potassium mM 1.8 0 0 40 5Phosphate CoSO₄•7H₂O g/L 0.0024 0.0024 0 0.0024 0.0048 CuSO₄•5H₂O g/L0.02 0.02 0.00 0.02 0.04 ZnSO₄•7H₂O g/L 0.0024 0.0024 0 0.0024 0.0048Na₂MoO₄•2H₂O g/L 0.0008 0.0008 0 0.0008 0.0016 H₃BO₃ g/L 0.0004 0.0004 00.0004 0.0008 MnSO₄•H₂O g/L 0.0012 0.0012 0 0.0012 0.0024 FeSO₄•7H₂O g/L0.022 0.022 0 0.022 0.044 MgSO₄ mM 2 2 0 2 2.5 CaSO₄ mM 0.05 0.05 0 0.050.06 Glucose g/L 45 25 0 45 25 MOPS mM 200 200 0 200 0 Thiamine-HCl g/L0.01 0.01 0 0.01 0.01

Micro-Fermentations

An overview of the micro-fermentation protocol is illustrated in FIG.16A-C. Strains were evaluated for production in 96 well platemicro-fermentations, wherein cells were initially grown to mid-logphase, harvested, washed, resuspended and normalized in a phosphate freeproduction medium to an OD600=1, for a 24 hour production stage. Thesuccess of the micro-fermentations required: (1) syncing strains up byharvesting all strains in exponential phase; (2) the use of low biomasslevels, so that batch sugar could be kept low while enabling significantpotential product accumulation; and (3) a method to supply adequatemixing and aeration, while minimizing evaporative losses. To address thefinal requirement, commercially available microplate sandwich covers andclamps from EnzyScreen™ as used, which greatly reduce evaporative losseswhile enabling high levels of mixing and aeration in standard 25 mmorbit shakers operating at 400 rpm⁹²⁻⁹³. Micro-fermentation results foralanine production with different insulated phosphate promoters areshown in FIG. 17. Micro-fermentation results for strains evaluated withgapA and gapN gene alterations are given in FIG. 18.

Section 8: Micro-Fermentations Robustness Evaluation

During micro-fermentation oxygen robustness studies, production culturevolume was varied to achieve desired oxygen transfer rate (OTR) valuesas previously reported(http://www.enzyscreen.com/oxygen_transfer_rates.htm)⁹²⁻⁹³ and as listedbelow in Table 14. Batch glucose levels during the production stage werealtered to assess robustness to glucose. Strains utilized in therobustness experiments at the micro-fermentation scale are listed inTable 15. Results from the micro-fermentation robustness studies aregiven in FIGS. 19A-D. FIGS. 20A-D, FIGS. 21A-D, FIGS. 22A-D, FIGS.23A-D, FIGS. 24A-D. FIGS. 25A-D, FIGS. 26A-D. FIGS. 27A-D, FIGS. 28A-D,FIGS. 29A-D. FIGS. 30A-D, FIGS. 31A-D, and FIG. 32.

TABLE 14 Culture conditions for different OTR values. 25 mm orbit shakerMax OTR Shaking Speed Fill Volume (mmol/L-hr) (rpm) (μL) 25 400 100 20400 150 15 400 200

TABLE 15 List of strains used for micro-fermentation robustnessevaluations and their RS scores. Strain # Silencing Proteolysis PlasmidRS  1 gltA1 FU pSMART-Ala2  89.6  2 gltA1 F pSMART-Ala2  89.5  3 gltA1GU pSMART-Ala2  89.4  4 FG1G2 None pSMART-Ala2  89.3  5 G1G2 GUpSMART-Ala2  88.8  6 FG1G2 G pSMART-Ala2  88.2  7 G1G2 F pSMART-Ala2 83.4  8 gltA2 FGU pSMART-Ala2  83.4  9 gltA1 FGU pSMART-Ala2  83.1 10G1G2 FGU pSMART-Ala2  82.3 11 gltA2 U pSMART-Ala2  82.2 12 gltA2 FpSMART-Ala2  80.6 13 FG1G2 FG pSMART-Ala2  80.5 14 None G pSMART-Ala2 79.9 15 gltA2 GU pSMART-Ala2  77.9 16 fabI FGU pSMART-Ala2  75.7 17 NoneFG pSMART-Ala2  75.4 18 G1G2 FU pSMART-Ala2  75.3 19 None FGUpSMART-Ala2  73.4 20 None FU pSMART-Ala2  73.3 21 gltA1 U pSMART-Ala2 72.9 22 fabI FG pSMART-Ala2  69.1 23 FG1G2 FU pSMART-Ala2  67.6 24 gltA2FU pSMART-Ala2  67.5 25 None F pSMART-Ala2  65.6 26 gltA2 FGpSMART-Ala2  67.1 27 FG1G2 F pSMART-Ala2  61.1 28 fabI GU pSMART-Ala2 59.9 29 fabI F pSMART-Ala2  59.6 30 gltA1 FG pSMART-Ala2  58.1 31 gltA1None pSMART-Ala2  57.1 32 None None pSMART-Ala2  55.5 33 G1G2 NonepSMART-Ala2  54.1 34 fabI U pSMART-Ala2  53.9 35 gltA2 G pSMART-Ala2 52.8 36 fabI None pSMART-Ala2  50.3 37 fabI FU pSMART-Ala2  48.4 38gltA2 None pSMART-Ala2  47.8 39 FG1G2 FGU pSMART-Ala2  44.6 40 None GUpSMART-Ala2  42.9 41 None U pSMART-Ala2  39.3 42 fabI G pSMART-Ala2 39.2 43 gltA1 G pSMART-Ala2  34.7 44 G1G2 FG pSMART-Ala2  32.8 45 FG1G2U pSMART-Ala2  29.4 46 FG1G2 GU pSMART-Ala2  24.3 47 G1G2 G pSMART-Ala2 24.1 48 G1G2 U pSMART-Ala2  −25.3 49 None None pSMART-Ala13 55.7 50 NoneNone pSMART-Ala12 −31.5 51 None None pSMART-Ala15 −103.2 52 None NonepSMART-Ala11 −114.1 53 None None pSMART-Ala14 −441.5

Section 9: Standardized 2-Stage Fermentation

A standardized phosphate limited 2-stage fermentation protocol wasutilized for evaluation of all valve strains. This protocol yieldshighly reproducible growth stage results, with minimal strain to strainvariability even with strains making different products. Moresignificant variability was observed during the production stage as aresult of differing feed rates and base utilization by differentstrains. FIG. 33A gives the growth curves for all valve strains with a10 g·cdw/L biomass level in 1 L fermentations performed in this study.This consistency is contrasted to the more variable growth of growthassociated production strains, given in FIG. 33B.

TABLE 16 Strains used for rnevalonic acid scalability. Strain #Silencing Proteolysis Plasmid 1 FG1G2 FU pSMART-Mev2 2 G2Z FGUApSMART-Mev2 3 FG1G2A FUN pSMART-Mev2 4 UZ FGUA pSMART-Mev2

Section 10: Analytical Methods

TABLE 17 UPLC-MS/MS parameters Retention ESI MRM Cone Collision AnalyteTime (min) Mode Transition(s) Voltage Energy Alanine 0.5 + 89.95 → 44.0815 9 C13-Alanine 0.5 + 91.95 → 46.06 15 9

DETAILED DESCRIPTION OF FIGURES

FIG. 1A: An Overview of Dynamic Metabolic Control in 2-StageFermentations. Metabolic engineering involves optimizing a metabolicpathway to a desired product to the existing metabolic network of ahost, converting feedstocks to a desired product. Filled circlesindicate metabolites and lines indicate enzymatic reactions. Traditionaloptimization in metabolic engineering, often involves three key steps(a) the deletion of competing non-essential metabolic pathways includingthose leading to undesired byproducts and the overexpression of enzymesin the pathway converting feedstock molecules to the product (indicatedby thicker lines) and potentially (b) attenuating enzymes in essentialmetabolism (indicated by orange lines) to further increase production.This process is iterated to optimize the yield to the desired product(pie charts). By contrast, dynamic metabolic network minimization can beused to fully unlock the potential of commonly used 2-stage fermentationprocesses (c-d). In the first stage of these processes (c) biomassgrowth and yield are optimized, while in the second stage (d) productformation is optimized, which is well suited for a 2-stage process (e)in which biomass levels accumulate and consume a limiting nutrient (inthis case inorganic phosphate), which when depleted triggers entry intoa productive stationary phase. Synthetic metabolic valves utilizingCRISPRi based gene silencing and/or controlled proteolysis can be used(f and g) to greatly reduce the pertinent metabolic network upon thetransition to the production stage, (f) and array of silencing guidescan be induced, processed by the CASCADE complex into individual guidesand used to silencing target multiple genes of interest (GOI). (g) IfC-terminal DAD+4 lags are added to enzymes of interest (EOI) throughchromosomal modification, they can be inducibly degraded by the clpXPprotease in the present of and inducible sspB chaperone. (h) Dynamiccontrol over protein levels in E. coli using 2 stage dynamic controlwith inducible proteolysis and CRISPRi silencing. As cells growphosphate is depleted, and cells “turn off mCherry and “turn on” GFPuv.Shaded areas represent one standard deviation from the mean, n=3. (i)Relative impact of proteolysis and gene silencing alone and incombination on mCherry degradation, with (j) decays rates.

FIG. 1B: Strain and Bioprocess Optimization. (a) Conventional approachesfor strain and process optimization in metabolic engineering ofteninvolves deletion of competing non-essential metabolic pathways andoverexpression of pathway enzymes (Filled circles: metabolites; lines:enzymatic reactions, green indicated a production pathway). (a-i) Strainvariants are evaluated at screening scale (microtiter plates, shakeflasks, etc), (a-ii) the best strains are assessed in larger scaleinstrumented bioreactors. Numerous design-build-test cycles (a-vi-vii)are used to iteratively optimize both the production strain and process,including the often-critical optimization of environmental (process)variables (a-vii). (a-iii) The best performing strains and associatedoptimized process conditions are scaled to industrially relevant levels.(b) Rapid strain and bioprocess optimization using 2-stage dynamicmetabolic control. The metabolic network in the cell is dynamicallyminimized to only the steps essential for product formation. This isaccomplished in a standardized 2-stage bioprocess (c), where a biomassaccumulating growth stage is followed by a production stage, with only aminimal metabolic network. The limitation of a macronutrient can be usedto “switch” cellular metabolism from growth to production. The approachresults in a smaller subset of potential strain variants for screening(b-i). Metabolic network minimization helps increase relevant metabolitelevels (d) and thus production levels, it also enhances processrobustness (e), and as a result process and strain scalability (f). Thebest producers identified from screening are predictably and rapidlyscaled to (b-ii) larger instrumented bioreactors, and (b-iii)subsequently to industrially relevant levels. If needed, limiteddesign-build-test cycles (b-iv) are incorporated to guide improvements.Product independent, standardized protocols are followed for strainevaluation at all scales, eliminating the need for intensive processoptimization.

FIGS. 2A-D: Implementation of 2-stage Synthetic Metabolic Valves (SMVs)in E. coli. FIG. 2A depicts SMVs utilizing CRISPRi based gene silencingand/or controlled proteolysis were constructed. (Top) Silencing: Anarray of inducible silencing guide RNAs (i) can be used to silenceexpression of multiple genes of interest (GOI) when the native E. coliCRISPR/Cascade machinery is expressed, which can process guide arraysinto individual guides (ii). (Bottom) Proteolysis: When C-terminal DAS+4tags are added to enzymes of interest (EOI) (through chromosomalmodification), they can be degraded by the clpXP protease (iv) upon thecontrolled induction of the sspB chaperone (iii). FIG. 2B depictsdynamic control over protein levels in E. coli using inducibleproteolysis and CRISPRi silencing. As cells grow phosphate is depleted,cells “turn OFF” mCherry and “turn ON” GFPuv. Shaded areas represent onestandard deviation from the mean, r.f.u, relative fluorescence units.FIG. 2C depicts relative impact of proteolysis and gene silencing aloneand in combination on mCherry degradation, n.f.u, normalizedfluorescence units (normalized to maximal fluorescence). FIG. 2D depictsrelative impact of proteolysis and gene silencing alone and incombination on observed mCherry fluorescence decays rates (per hour).

FIGS. 3A-K: Alanine Production in E. coli utilizing 2-stage DynamicControl. FIG. 3A depicts strain variant design. Primary pathways incentral metabolism are shown including: Glycolysis, the PentosePhosphate Pathway, the Citric Acid Cycle (TCA), Fatty Acid Biosynthesis,and the Soluble Transhydrogenase. Key valve candidate enzymes/genes thatare “turned OFF” to reduce flux through central metabolism can include:glucose-6-phosphate dehydrogenase (zwf-“Z”), lipoamide dehydrogenase(lpd-“L”), citrate synthase (gltA-“G”), enoyl-ACP reductase (fabI-“F”),and the soluble transhydrogenase (udhA-“U”). Importantly, dynamicelimination of fabI has been previously demonstrated to increaseintracellular malonyl-CoA pools as well as malonyl-CoA flux⁵⁵. Enzymesthat are dynamically “turned ON” can include the metabolic pathways toproduce the products of interest, in this case alanine. Specific pathwayenzymes include an NADPH-dependent alanine dehydrogenase (ald*) and analanine exporter (alaE). Additionally, as the alanine production pathwayutilizes NADPH as a cofactor, the NADPH-dependentglyceraldehyde-3-phosphate dehydrogenase encoded by the gapN gene⁵⁶ fromS. mutans was turned on alone and in combination with turning off thenative gapA-“A” gene (NADH dependent glyceraldehyde dehydrogenase).Abbreviation: PTS—glucose phosphotransferase transport system,P—phosphate, BP—bisphosphate, OAA—oxaloacetate, DHAP—dihydroxyacetonephosphate, GA3P—glyceraldehyde-3-phosphate, 1,3-BPG—1,3bisphosphoglycerate, 3-PG—3-phosphoglycerate, 2-PG—2-phosphoglycerate.PEP—phosphoenolpyruvate, MSA—malonate semialdehyde, ACP acyl carrierprotein, Ru—ribulose, Xu—xylulose, E—erthryose, Ri—ribose,S—sedoheptulose. Strains were engineered with SMVs for the dynamiccontrol of all combinations of valve genes/enzymes, either through genesilencing alone, proteolysis alone, or the combination of both. Thesestrains were evaluated for alanine production in standardizedmicro-fermentations. FIG. 3B depicts rank order plot for average alaninetiter (black) of all valve strains examined in 2-stagemicro-fermentation, grey area represents standard deviation. Alanineproduction in the control strain was colored in red. FIG. 3C depictsaverage alanine titer in 2-stage production in response to differentproteolysis and silencing combinations, from 0 g/L (purple) to 5 g/L(red). FIG. 3D depicts average alanine titer in response to differentoxygen transfer rates (OTR) and glucose concentrations evaluated for asingle “Valve” alanine strain (Silencing of gltA1 (“G”), Proteolysis offabI and udhA (“FU”)). The results of this surface were used tocalculate a strain-specific robustness score (RS) (refer to text), thisstrain has the highest RS score. FIG. 3E depicts a heat map of therobustness score for a subset of 48 “Valve” strains evaluated acrossmultiple process conditions. FIG. 3F depicts scale up of one of the bestproducing strain from micro-fermentations (Silencing of fabI-gltA1-gltA2(“FG1G2”), Proteolysis of fabI, gltA and udhA (“FGU”)) to 1 Lbioreactors results in a titer of 80 g/L after 48 hrs of production,with a yield of 0.8 gig. FIG. 3G depicts overexpression of the alaEalanine exporter in this strain (Panel f) results in significantlyimproved production, reaching 147 g/L in 27 hrs of production, with ayield of ˜1 g/g. (Refer to Supplemental Materials, Section 3 foradditional details). FIG. 3H depicts strains selected for robustnessevaluation in micro-fermentations. FIG. 31 depicts robustness and titerfor the most robust “Valve” alanine strain (Silencing_gltA1,Proteolysis_FU). Bottom surface shows heat map for the alanine titernormalized to the median of all process conditions assessed, uppersurface shows alanine tiler under all process conditions, the same colorscale (alanine titer in g/L) was used for both panels. FIG. 3J depictsRS3 scores for the selected strains. FIG. 3K depicts processreproducibility heat map for all conditions evaluated, the samegrayscale was used for FIG. 3J and FIG. 3K.

FIGS. 4A-F: Robustness Comparison Between 2-Stage and Growth AssociatedApproaches. FIG. 4A depicts rank order of the RS3 scores for all alaninestrains evaluated, red bars indicate valve alanine strains, and bluebars indicate growth associated (GA) alanine strains. FIG. 4B depictsaverage RS3 score for “Valve” alanine strains with proteolysis “F”valve, and growth associated alanine strains. FIG. 4C depicts max titerplot for a representative “Valve” alanine (Proteolysis_FGU,Silencing_gltA1), and growth associated alanine strains inmicro-fermentation of all conditions evaluated. FIG. 4D depicts processreproducibility for growth associated alanine strains under allconditions evaluated. FIG. 4E depicts robustness and titer for arepresentative robust “Valve” alanine (Proteolysis_FGU,Silencing_gltA1). FIG. 4F depicts robustness and titer for the GA2strain. Bottom surface, heat map for the alanine titer normalized to themedian of all process conditions assessed, upper surface, alanine titerunder all process conditions, the same color scale (alanine titer ing/L) was used for both panels.

FIGS. 5A-J: Comparisons of “Valve” and growth associated alanineproduction in micro-fermentations (FIGS. 5A-D) and 1 L fermentation(FIGS. 5E-J). Average alanine titer (FIG. 5A) and robustness score (FIG.5B) for all strains used for robustness analysis. Average alanine titerin response to different OTR and glucose concentrations for selected“Valve” (FIG. 5C) and growth associated (FIG. 5D) alanine strains.Strains marked by asterisk in (FIG. 5B) were used for this analysis.These two strains were selected for 1 L performance comparison. FigureSE and Figure SF depicts 1 L performance metrics evaluated, includingaverage specific productivity (SP, g/gdcw-h), average glucose uptakerate (GUR, g/gcdw-h), max titer (g/L), and max yield (gig). Figure G andFigure H depicts μL to 1 L scalability. 1 L data was standardized to themaximal titer within 50 hours of production. Adequate feed was used forgrowth associated strains to avoid glucose depletion. FIG. 5I and FIG.5J depicts 1 L production profiles for all strains used in scalabilityplot FIG. 5G and FIG. 5H respectively, darker symbols represent growthcurves, lighter symbols represent production curves, shape of symbolsencode the same strains in FIG. 5G or FIG. 5H.

FIG. 6A-E: Mevalonate Production in E. coli utilizing 2-stage DynamicControl. FIG. 6A depicts Metabolic Pathways and SMVs for mevalonateproduction. FIG. 6B depicts mevalonate production using severalproduction pathway plasmid variants with varied promoter combinations inthe control strain. FIG. 6C depicts micro-fermentation results for asubset of “Valve” strains producing mevalonate, using the bestproduction pathway from FIG. 6B, along with combinations of proteolyticand silencing SMVs. FIG. 6D depicts μL to 1 L scalability for a subsetof mevalonate strains evaluated at the 1 L scale, n=3 for μL data andn=1 for 1 L data. The maximal liter within 50 hours of production timewas used for the correlation. FIG. 6E depicts production of the bestmevalonate strain from FIG. 6D (Silencing of fabI-gltA1-gltA2 (“FG1G2”).Proteolysis of fabI and udhA (“FU”)) in 1 L bioreactors. A titer of 97g/L was observed in 78 hrs of production. Yields during the productionstage reached 0.46 g/g (84% of theoretical yield). (Refer toSupplemental Materials, Section 9 for additional details). FIG. 6Fdepicts micro-fermentation results for a subset of strains producing3-HP. FIG. 6G depicts μL to 1 L scalability for a subset of 3-HP strainsevaluated at the 1 L scale (Supplemental Materials Tables S21 and S22).FIG. 6H depicts production performance for the best 3-HP strains in the1 L systems, squares, 3-HP/mevalonic acid titer; circles, OD600. Yieldsduring the production stage reached for the 0.46 g/g for mevalonic acidand 0.63 g/g for 3-HP in the highest producers.

FIG. 7: Phosphate depletion promoter characterization. A set of GFPreporter vectors were constructed to assess the expression level of 12previously identified phosphate regulated promoters. Strains wereevaluated continuously for GFP expression in the Biolector™ using astandardized protocol wherein in minimal medium limited for phosphate isused. After Biomass levels reach a peak (not shown for clarity). GFPexpression begins. Importantly the current set of promoters enables alarge range of expression levels.

FIG. 8: Insulated phosphate depletion promoter characterization. A setof GFP reporter vectors were constructed to assess the expression levelof five insulated phosphate regulated promoters in FGM3 media. Strainswere evaluated continuously for GFP expression in the Biolector™ using astandardized protocol wherein in minimal medium limited for phosphate isused. After Biomass levels reach a peak (not shown for clarity). GFPexpression begins. Importantly the current set of promoters enables alarge range of expression levels.

FIG. 9: Insulated constitutive promoter characterization. A set of GFPreporter vectors were constructed to assess the expression level of fiveinsulated constitutive promoters in FGM3 with 40 mM phosphate media.Shaded area represents standard deviations, n=3. Strains were evaluatedcontinuously for GFP expression in the Biolector™. GFP expression n asobserved only for promoters proA, proB and proD.

FIG. 10: Metabolic modeling results for optimal 3-HP flux in two stagefermentations. LEFT: Optimized fluxes during the growth stage wherebiomass production was used as the objective function. RIGHT: Optimizedfluxes during the 3-HP production stage where 3-HP production was usedas the objective function (biomass production was set to 0). Fluxes arelisted as relative ratios or moles of flux through a given reaction per100 moles of glucose utilized.

FIG. 11: Chromosomal modifications.

FIG. 12: Average maximal growth rates of starting host strains in 1 LFGM10 minimal medium fermentations, n=2.

FIG. 13A-E: Distribution of glucose utilized during the growth phase ofstarting host strains in 1 L standard minimal medium fermentations. Midexponential and final growth period results are given for DLF 0025 as“production” begins in mid-late exponential phase. Results are averagesof duplicate fermentations. FIG. 13A. BW25113; FIG. 13B. BWapldf; FIG.13C, DLF_0001; FIG. 13D, DLF_0025 at mid-exponential; FIG. 13E, DLF 0025at end of growth phase. Unit was gram glucose.

FIG. 14: pCASCADE-control plasmid construction scheme.

FIG. 15A-B: pCASCADE construction scheme. FIG. 15A, single sgRNAcloning; FIG. 15B, double sgRNA.

FIG. 16A-C: Micro-fermentation process overview. (A) An overview of thehigh throughput micro-fermentation protocol. Freezer stocks(alternatively colonies may be used) are used to inoculate into SM10++in 96 well plates. Cultures are grown overnight for 16 hours, harvestedby centrifugation, washed with no-phosphate medium and resuspended inno-phosphate medium at target biomass levels. (OD600 nm=1.0).EnzyScreen™ covers and clamps are used to reduce evaporation and enablehigh oxygen transfer rates. The protocol is implemented with a Tecan Evoliquid handler. (B) Representative overnight growth in a 96 well platesculture, distribution of OD600 for overnight culture was plotted. (C)Representative OD600 distribution after normalization using Tecan Evoliquid handler.

FIG. 17: Micro-fermentation for L-alanine production using differentinsulated phosphate promoters in DLF_0025 strain.

FIG. 18: Heatmap for L-alanine production by gapN/gapA strains.

FIGS. 19A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 20A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 21A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 22A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 23A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 24A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 25A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 26A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 27A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 28A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 29A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 30A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIGS. 31A-D: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for 4 strains evaluated forrobustness.

FIG. 32: Alanine production in response to different OTR and glucoseconcentration in micro-fermentation for one strain evaluated forrobustness.

FIGS. 33A-B: Growth profile for all (FIG. 33A) valve and (FIG. 33B)growth associated strains at 1 L scale evaluated in this paper. Growthcurves were synced to account for any variations in lag time. Valvestrains growth curves were synced to the same mid-exponential point.Growth associated strains growth curves were synced to the same take-offpoint.

FIG. 34: Specific Productivity (SP) comparison for strain with highestmevalonate titer from literature and mevalonate strain 1 evaluated inthis work.

FIG. 35: Alanine standard curve from MS measurement. Average andstandard deviation for mass spec response from triplicate standardmeasurement were plotted.

FIGS. 36A-B: Glucose (FIG. 36A) and ethanol (FIG. 368) standard curvesfrom RI measurement. Average and standard deviation for peak area fromtriplicate standard measurement were plotted.

FIG. 37: 3-Hydroxypropionic acid standard curve from TUV measurement.Average and standard deviation for peak area from duplicate standardmeasurement were plotted.

FIGS. 38A-D: TUV standard curves for (FIG. 38A) L-alanine, (FIG. 38B)D-alanine, (FIG. 38C) mevalonic acid, and (FIG. 38D) mevalonolactone.Average and standard deviation for peak area from triplicate standardmeasurement were plotted.

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While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the an thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-106. (canceled)
 107. A multi-stage fermentation bioprocess forproducing a product from a genetically modified microorganism,comprising: providing a genetically modified microorganism having aproduction pathway for producing a product that is: an amino acid,acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters,isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene,isobutylene, ethyl acetate, vinyl acetate, 1,4-butanediol,2,3-butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate,2-OH-isobutyrate, 3-OH-butyrate, ethanol, isopropanol, D-lactate,L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate,caprolactam, adipic acid, propanol, isopropanol, fused alcohols,1,2-propanediol, 1,3-propanediol, formate, fumaric acid, propionic acid,succinic acid, valeric acid, maleic acid, or poly-hydroxybutyrate;growing the identified genetically modified microorganism in a media ina growth phase, the genetically modified microorganism comprising: i. aproduction pathway comprising at least one production enzyme forbiosynthesis of the product; and ii. one or more synthetic metabolicvalves for reducing or eliminating flux through multiple metabolicpathways within the genetically modified microorganism when thesynthetic metabolic valves are induced, the one or more syntheticmetabolic valves comprising: a) at least one silencing syntheticmetabolic valve that silences gene expression of a gene selected from:fabI, gltA, lpd, zwf, and udhA, or b) at least one proteolytic syntheticmetabolic valve that controls proteolysis of a proteolyzable enzymeselected from: fabI, gltA, lpd, zwf, and udhA; transitioning to aproductive stationary phase, the transition comprising: depletion of alimiting nutrient; inducing the one or more synthetic metabolic valves;activation of the production pathway; and producing the product. 108.The multi-stage fermentation bioprocess of claim 107, wherein themicroorganism comprises a silencing synthetic metabolic valve and aproteolytic synthetic metabolic valve, and wherein the activation of thesilencing synthetic metabolic valve produces a product that enhances thefunction of the proteolytic synthetic metabolic valve.
 109. Themulti-stage fermentation bioprocess of claim 107, wherein themicroorganism comprises a chromosomal a deletion or disruption of a cas3or sspB gene.
 110. The multi-stage fermentation bioprocess of claim 107,wherein the rate of production of said product during the productivestationary phase is reduced less in response to a change of anenvironmental condition as compared to a cell lacking the syntheticmetabolic valves.
 111. The multi-stage fermentation bioprocess of claim107, wherein the silencing synthetic metabolic valve silences geneexpression of a gene selected from: fabI, gltA, lpd, zwf, and udhA andan additional gene.
 112. The multi-stage fermentation bioprocess ofclaim 107, wherein the proteolytic synthetic metabolic valve thatcontrols proteolysis of a proteolyzable enzyme selected from: fabI,gltA, lpd, zwf, and udhA and an additional enzyme.
 113. The multi-stagefermentation bioprocess of claim 107, wherein at least one silencingsynthetic metabolic valve is characterized by CRISPR interference ofgene expression of a gene that is a fabI, gltA, lpd, zwf, or udhA geneand expression of a CASCADE plasmid comprising an array of guide RNAgenes.
 114. The multi-stage fermentation bioprocess of claim 107,wherein at least one proteolytic synthetic metabolic valve ischaracterized by expression of the proteolytic enzyme operably linked toa C-terminal DAS4 peptide tag and controlled proteolysis of a fabI,gltA, ldp, zwf, or udhA enzyme by the synthetic metabolic valve isselective for the tag by clpXP protease upon induction of sspB chaperoneprotein.
 115. The multi-stage fermentation bioprocess of claim 107,wherein the microorganism has reduced level or activity of at least onemetabolic enzyme prior to synthetic metabolic valve induction.
 116. Agenetically modified microorganism comprising: i. a production pathwaycomprising at least one production enzyme for biosynthesis of a productselected from the group: an amino acid, acetate, acetoin, acetone,acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol,ethylene glycol, ethylene, propylene, butylene, isobutylene, ethylacetate, vinyl acetate, 1,4-butanediol, 2,3-butanediol, butanol,isobutanol, sec-butanol, butyrate, isobutyrate, 2-OH-isobutyrate,3-OH-butyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate,itaconate, levulinate, glucarate, glutarate, caprolactam, adipic acid,propanol, isopropanol, fused alcohols, 1,2-propanediol, 1,3-propanediol,formate, fumaric acid, propionic acid, succinic acid, valeric acid,maleic acid, or poly-hydroxybutyrate; and ii. one or more syntheticmetabolic valves for reducing or eliminating flux through multiplemetabolic pathways within the genetically modified microorganism whenthe synthetic metabolic valves are induced, the one or more syntheticmetabolic valves comprising: a) at least one silencing syntheticmetabolic valve that silences gene expression of a gene selected from:fabI, gltA, lpd, zwf, and udhA, or b) at least one proteolytic syntheticmetabolic valve that controls proteolysis of a proteolyzable enzymeselected from: fabI, gltA, lpd, zwf, and udhA; and wherein growth of thegenetically modified microorganism is slowed or stopped and productproduction is enhanced, as compared to a microorganism lacking theproduction pathway or synthetic metabolic valve, by inducing thesynthetic metabolic valve, and wherein growth of the geneticallymodified microorganism is slowed or stopped by depletion of a limitingnutrient thereby inducing a stationary phase, and product production isenhanced, as compared to a microorganism lacking the production pathwayor synthetic metabolic valve, by inducing the synthetic metabolic valvein the stationary phase.
 117. A multi-stage fermentation bioprocess forproducing a product from a genetically modified E. coli, comprising:providing a genetically modified E. coli having a production pathway forproducing a product that is: an amino acid, acetate, acetoin, acetone,acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol,ethylene glycol, ethylene, propylene, butylene, isobutylene, ethylacetate, vinyl acetate, 1,4-butanediol, 2,3-butanediol, butanol,isobutanol, sec-butanol, butyrate, isobutyrate, 2-OH-isobutyrate,3-OH-butyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate,itaconate, levulinate, glucarate, glutarate, caprolactam, adipic acid,propanol, isopropanol, fused alcohols, 1,2-propanediol, 1,3-propanediol,formate, fumaric acid, propionic acid, succinic acid, valeric acid,maleic acid, or poly-hydroxybutyrate; growing the identified geneticallymodified E. coli in a media in a growth phase, the genetically modifiedE. coli comprising: i. a production pathway comprising at least oneproduction enzyme for biosynthesis of the product; and ii. one or moresynthetic metabolic valves for reducing or eliminating flux throughmultiple metabolic pathways within the genetically modified E. coli whenthe synthetic metabolic valves are induced, the one or more syntheticmetabolic valves comprising: a) at least one silencing syntheticmetabolic valve that silences gene expression of a gene, or b) at leastone proteolytic synthetic metabolic valve that controls proteolysis of aproteolyzable enzyme; transitioning to a productive stationary phase,the transition comprising: depletion of a limiting nutrient; inducingthe one or more synthetic metabolic valves; and activation of theproduction pathway; and producing the product.
 118. The multi-stagefermentation bioprocess of claim 117, wherein the silencing syntheticmetabolic valve that silences gene expression of a gene is a geneselected from the group: fabI, gltA, ldp, zwf, or udhA.
 119. Themulti-stage fermentation bioprocess of claim 117, wherein the silencingsynthetic metabolic valve silences gene expression of a gene selectedfrom: fabI, gltA, lpd, zwf, and udhA and an additional gene.
 120. Themulti-stage fermentation bioprocess of claim 117, wherein theproteolytic synthetic metabolic valve that controls proteolysis of aproteolyzable enzyme is an enzyme selected from the group: fabI, gltA,ldp, zwf, or udhA.
 121. The multi-stage fermentation bioprocess of claim117, wherein the proteolytic synthetic metabolic valve that controlsproteolysis of a proteolyzable enzyme selected from: fabI, gltA, lpd,zwf, and udhA and an additional enzyme.
 122. The multi-stagefermentation bioprocess of claim 117, wherein at least one silencingsynthetic metabolic valve is characterized by CRISPR interference ofgene expression of a gene that is a fabI, gltA, lpd, zwf, or udhA geneand expression of a CASCADE plasmid comprising an array of guide RNAgenes.
 123. The multi-stage fermentation bioprocess of claim 117,wherein at least one proteolytic synthetic metabolic valve ischaracterized by expression of the proteolytic enzyme operably linked toa C-terminal DAS4 peptide tag and controlled proteolysis of a fabI,gltA, ldp, zwf, or udhA enzyme by the synthetic metabolic valve isselective for the tag by clpXP protease upon induction of sspB chaperoneprotein.
 124. The multi-stage fermentation bioprocess of claim 117,wherein the rate of production of said product during the productivestationary phase is reduced less in response to a change of anenvironmental condition as compared to a cell lacking the syntheticmetabolic valves.
 125. The genetically modified E. coli of claim 117,comprising: i. a production pathway comprising at least one productionenzyme for biosynthesis of a product selected from the group: an aminoacid, acetate, acetoin, acetone, acrylic, malate, fatty acid ethylesters, isoprenoids, glycerol, ethylene glycol, ethylene, propylene,butylene, isobutylene, ethyl acetate, vinyl acetate, 1,4-butanediol,2,3-butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate,2-OH-isobutyrate, 3-OH-butyrate, ethanol, isopropanol, D-lactate,L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate,caprolactam, adipic acid, propanol, isopropanol, fused alcohols,1,2-propanediol, 1,3-propanediol, formate, fumaric acid, propionic acid,succinic acid, valeric acid, maleic acid, or poly-hydroxybutyrate; andii. one or more synthetic metabolic valves for reducing or eliminatingflux through multiple metabolic pathways within the genetically modifiedE. coli when the one or more synthetic metabolic valves are induced, theone or more synthetic metabolic valves comprising: a) at least onesilencing synthetic metabolic valve that silences gene expression of agene encoding at least one silenceable enzyme, or b) at least oneproteolytic synthetic metabolic valve that controls proteolysis of aproteolyzable enzyme; and wherein growth of the genetically modified E.coli is slowed or stopped and product production is enhanced, ascompared to E. coli lacking the production pathway or syntheticmetabolic valve, by inducing the synthetic metabolic valve, and whereingrowth of the genetically modified microorganism is slowed or stopped bydepletion of a limiting nutrient thereby inducing a stationary phase,and product production is enhanced, as compared to a microorganismlacking the production pathway or synthetic metabolic valve, by inducingthe synthetic metabolic valve in the stationary phase.
 126. Themicroorganism of claim 125, wherein the silencing synthetic metabolicvalve that silences gene expression of a gene is a gene selected fromthe group: fabI, gltA, ldp, zwf, or udhA; or the proteolytic syntheticmetabolic valve that controls proteolysis of a proteolyzable enzyme isan enzyme selected from the group: fabI, gltA, ldp, zwf, or udhA.